Nine-methine dye probes and their use in disease diagnosis

Abstract

The application belongs to the technical field of small molecule fluorescent probe, and provides a kind of nonamethylenecyclooctyne dye probe and its application in disease diagnosis.A kind of nonamethylenecyclooctyne dye is prepared, and the dye can be rapidly covalently combined with serum albumin in vivo or in vitro to form fluorescent protein, which is suitable for targeted imaging of various diseases.Nonamethylenecyclooctyne dye is injected through tail vein, rapidly combined with serum albumin in vivo, and can be used for targeted imaging and diagnosis of different degrees of brain damage in mouse stroke and targeted imaging of tumor.In addition, the pre-prepared nonamethylenecyclooctyne dye and human serum albumin complex can also be injected through tail vein to achieve rapid targeted imaging of tumor.Compared with ICG, NIR-940 can rapidly covalently combine with serum albumin at 37 DEG C or even room temperature, overcoming the defect of non-covalent combination of ICG, and being suitable for high-contrast targeted imaging of disease area.

Description

Technical Field

[0001] This invention belongs to the field of small molecule fluorescent probe technology, and particularly relates to a class of nonamethyst dye probes and their application in disease diagnosis. Background Technology

[0002] Endogenous or exogenous serum albumin can non-specifically target various disease areas, making it a crucial and versatile biomarker. For example, ischemic stroke, an acute cerebrovascular disease, severely impairs the integrity of the blood-brain barrier (BBB), leading to increased permeability and allowing serum albumin leakage. Furthermore, cancer tissues efficiently express various albumin-binding proteins, such as cysteine-rich acidic secretory protein (SPARC) and glycoprotein 60 (gp60). Combined with the enhanced permeability and retention effect (EPR) of tumor tissue, this allows serum albumin to enter and remain in tumor tissue for extended periods.

[0003] Near-infrared II (NIIR) fluorescence in vivo imaging technology has been widely applied in various biomedical scenarios and has shown great potential for clinical translation. However, there is still a lack of an effective NIIR fluorescent probe that can covalently bind to albumin for high-contrast imaging and diagnosis of various diseases. Although indocyanine green (ICG), which has been approved by the FDA, can non-covalently bind to serum albumin, its binding efficiency is low, making it difficult to effectively target disease areas through albumin.

[0004] To address the aforementioned issues and further advance the application of near-infrared imaging technology in disease diagnosis, there is an urgent need to synthesize a novel near-infrared II serum albumin-targeting probe. Therefore, this invention proposes a class of nonamethystine dye probes and their application in disease diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide a type of nonamethyst dye probe and its application in disease diagnosis, aiming to solve the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A class of nonamethyst dye probes, for the nonamethyst dye NIR-940 with the following structure:

[0008]

[0009] Or dyes that are analogs of the nonamethyne molecular structure conforming to the following general formula I:

[0010] General Formula I:

[0011] NIR-940 or a nonamethyne dye molecule conforming to general formula I is covalently bound in situ to serum albumin in vivo; NIR-940 or a nonamethyne dye molecule conforming to general formula I is incubated with human serum albumin in vitro under heating conditions to obtain a covalent complex NIR-940@HSA.

[0012] Furthermore, the preparation process of the NIR-940 and the working solution of NIR-940@HSA is as follows:

[0013] Step a. Dissolve NIR-940 in DMSO to form a NIR-940 stock solution with a concentration of 20 mmol / L;

[0014] Step b. Dissolve 5 μL of NIR-940 stock solution directly in 200 μL of PBS to obtain a working solution of NIR-940 with a concentration of 500 μmol / L;

[0015] Step c. Dissolve human serum albumin solid in PBS to obtain a protein solution with a concentration of 10 μmol / L;

[0016] Step d. Add 5 μL of NIR-940 stock solution to 10 mL of protein solution, mix thoroughly by shaking, and then incubate in an oven at 50 °C for 2 h.

[0017] Step e. Concentrate the mixed solution obtained in step d to 200 μL using an ultrafiltration centrifuge tube at a speed of 8000-10000 rpm to obtain a NIR-940@HSA working solution with a concentration of 500 μmol / L.

[0018] Furthermore, in the NIR-940@HSA working solution, the molar ratio of NIR-940 to human serum albumin is 1:1.

[0019] Furthermore, the preparation steps of the NIR-940 are as follows:

[0020] Preparation of the linker: A mixture of N,N-dimethylformamide and dichloromethane was added to a flask dried in a high-temperature oven and equipped with a magnetic stir bar; the flask was purged with argon and cooled to 0°C; phosphorus oxychloride was slowly added while stirring at 0°C for 30 min; then decahydronaphthalene-1,8-dione was added dropwise; the ice bath was removed, and the reaction mixture was heated to 90°C and reacted for 3 h; the reaction mixture was poured onto crushed ice and cooled, and the product was extracted with dichloromethane; the combined organic layers were dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a thick orange liquid;

[0021] Preparation of NIR-940: 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-bromide, a linker, and 2,6-di-tert-butyl-4-methylpyridine were added to a flask dried in a high-temperature oven and dissolved in a mixture of n-butanol and toluene. The mixture was subjected to a freeze-vacuum-thaw cycle three times, and then heated to 100°C and held for 30 min. The crude product was directly purified by reversed-phase chromatography and then lyophilized to obtain NIR-940.

[0022] An application of the nonamethystine dye probe described above in the preparation of tumor-targeted imaging diagnostic reagents, wherein the reagent is directly used for imaging via tail vein injection during vascular imaging.

[0023] An application of the nonamethystine dye probe described above in the preparation of a targeted imaging diagnostic reagent for the brain of stroke-affected mice, wherein the reagent is directly used for imaging via tail vein injection.

[0024] An application of the nonamethystine dye probe described above in the preparation of a targeted imaging diagnostic reagent for skin lesions, wherein the reagent is directly used for imaging via tail vein injection.

[0025] An application of the nonamethystine dye probe described above in the preparation of a targeted imaging diagnostic reagent for blood-testis barrier disruption, wherein the reagent is directly used for imaging via tail vein injection.

[0026] An application of the nonamethystine dye probe described above in the preparation of a targeted imaging diagnostic reagent for intestinal vascular barrier disruption, wherein the reagent is directly used for imaging via tail vein injection.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention prepares a class of nonamethystine dyes that can rapidly covalently bind to serum albumin in vivo or in vitro to form fluorescent proteins, suitable for targeted imaging of various diseases. By injecting the nonamethystine dye via the tail vein, it rapidly binds to serum albumin in vivo, enabling targeted imaging and diagnosis of different degrees of brain damage in mice with stroke, as well as targeted imaging of tumors. Furthermore, a nonamethystine dye-human serum albumin complex prepared in vitro can also be injected via the tail vein for rapid targeted tumor imaging. Compared to ICG, NIR-940 can rapidly covalently bind to serum albumin at 37°C or even room temperature, overcoming the non-covalent binding limitation of ICG, making it suitable for high-contrast targeted imaging of disease areas. Attached Figure Description

[0029] Figure 1The absorption, fluorescence emission, and brightness of NIR-940 in dimethyl sulfoxide, mouse serum, and phosphate buffered aqueous solution are compared. Specifically: a) shows the absorption spectra of NIR-940 in dimethyl sulfoxide, mouse serum, and phosphate buffered aqueous solution; b) shows the fluorescence emission spectra of NIR-940 in dimethyl sulfoxide, mouse serum, and phosphate buffered aqueous solution; c) shows a comparison of the brightness of NIR-940 in dimethyl sulfoxide, mouse serum, and phosphate buffered aqueous solution.

[0030] Figure 2 This study analyzed the binding of NIR-940 to mouse serum albumin in vivo and in vitro. Specifically: a) gel electrophoresis results of mouse serum collected at different time points after intravenous injection of 500 μmol / L NIR-940; b) gel electrophoresis results of in vitro incubation at 37°C for 2 hours with different concentrations of mouse serum (dye and protein ratios from 1:1 to 1:25) ranging from 30 μmol / L to 30 μmol / L; c) [further details needed]. Figure 2 A statistical graph of the fluorescence intensity of the free dye bands on the gel electrophoresis bands obtained in b.

[0031] Figure 3 The results show the gel electrophoresis of NIR-940 and human serum albumin under different incubation conditions, the fluorescence intensity of the fluorescent protein bands, and the fluorescence intensity of the free dye bands; where: a) are the gel electrophoresis results of 10 μmol / L NIR-940 and 10 μmol / L human serum albumin in vitro after incubation at different temperatures for 2 h; b) are the gel electrophoresis results of 5-50 μmol / L NIR-940 and 10 μmol / L human serum albumin (protein and dye concentrations from 1:0.5 to 1:5) in vitro after incubation at 50°C for 2 h; c) are the gel electrophoresis results of 10 μmol / L NIR-940 and 10 μmol / L human serum albumin in vitro after incubation at 50°C for different times; d) are... Figure 3 The fluorescence intensity of fluorescent protein bands and free dye bands under different incubation conditions in AC.

[0032] Figure 4 High-resolution mass spectrometry analysis results of human serum albumin and NIR-940@HSA; where: a) is the high-resolution mass spectrometry result of human serum albumin (HSA); b) is the high-resolution mass spectrometry result of NIR-940@HSA obtained after incubating 10 μmol / L NIR-940 and 10 μmol / L human serum albumin in vitro at 50°C for 2 h.

[0033] Figure 5This diagram illustrates the use of NIR-940@HSA (obtained by covalently binding NIR-940 to exogenous human serum albumin in vitro) for rapid tumor-targeted imaging, and the use of NIR-940 directly via intravenous injection to covalently bind to endogenous mouse albumin in serum for tumor imaging or targeted imaging of the brains of stroke-affected mice.

[0034] Figure 6 Analysis of targeted imaging results of NIR-940 on the brains of mice with different degrees of stroke; where: a) is the targeted imaging result of NIR-940 administered directly via tail vein injection to the brains of mice with different degrees of stroke at different time points; b) is... Figure 6 The fluorescence intensity changes over time in stroke regions of different severity in region a; c is... Figure 6 The ratio of fluorescence signal to background signal in stroke regions of different severity in region a changes over time; d is... Figure 6 The change of fluorescence-labeled lesion area over time in stroke regions of different severities in type a.

[0035] Figure 7 This study analyzes the whole-body imaging and fluorescence signal changes in key tissues of mice bearing 4T1 tumors using NIR-940 and NIR-940@HSA, respectively. Specifically: a) whole-body imaging of mice bearing 4T1 tumors after subcutaneous xenografting of NIR-940 via tail vein injection in supine and lateral positions at different time points; b) whole-body imaging of mice bearing 4T1 tumors after subcutaneous xenografting of NIR-940@HSA via tail vein injection in supine and lateral positions at different time points; c) ... Figure 7 Changes in tumor, liver, and skin signals in mice over time in b; d is... Figure 7 a) Changes in tumor, liver, and skin signal intensity over time in mice; e) Changes in the tumor-liver signal ratio and tumor-skin signal ratio over time after injection of NIR-940 and NIR-940@HSA, respectively.

[0036] Figure 8 Analysis of long-term imaging monitoring results of NIR-940 on the skin and scalp injury areas on the back of mice; where: a) NIR-940, administered intravenously to mice with skin injuries on the back, enabled long-term imaging monitoring of the injury areas on the back; b) ... Figure 8 The corresponding signal-to-noise ratio of a; c is Figure 8 a represents the corresponding leakage area; d represents the long-term imaging monitoring of the scalp injury area by intravenous injection of NIR-940 into mice with scalp injuries; e represents... Figure 8 The corresponding signal-to-noise ratio of d; f is Figure 8 The corresponding leakage area of ​​d.

[0037] Figure 9This study analyzed the in vivo imaging and fluorescence signal changes in the damaged areas of mice with disrupted blood-testis barrier and intestinal vascular barrier using NIR-940. Specifically: a) local imaging of the damaged areas in mice intravenously injected with NIR-940 in the blood-testis barrier disrupted group and the control group; b) the change in signal-to-noise ratio (SNR) over time in the blood-testis barrier disrupted group and the control group; c) the change in fluorescence intensity in the testis region over time; d) local imaging of the intestinal region in mice intravenously injected with NIR-940 in the intestinal vascular barrier disrupted group and the control group; e) the change in SNR over time in the intestinal vascular barrier disrupted group and the control group; and f) the change in fluorescence intensity over time in the intestinal region.

[0038] Figure 10 This is the 1H NMR spectrum of NIR-920.

[0039] Figure 11 This is the 1H NMR spectrum of NIR-940. Detailed Implementation

[0040] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0041] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0042] One embodiment of the present invention provides a method for preparing a class of nonamethyst dyes, comprising the following steps:

[0043] Synthesize the nonamethyst dyes NIR-920 and NIR-940 with the following structures:

[0044]

[0045] The preparation methods for nonamethyst dyes NIR-920 and NIR-940 are as follows:

[0046] 1) Preparation of the linker;

[0047] The synthetic route for the linker is as follows:

[0048]

[0049] Synthesis of the linker: A mixture of N,N-dimethylformamide (DMF) (1.3 mL, 17 mmol, 5.4 equivalences) and dichloromethane (CH2Cl2) (0.5 mL) was added to a flask dried in a high-temperature oven and equipped with a magnetic stir bar. The flask was purged with argon and cooled to 0 °C. Phosphorus oxychloride (POCl3) (2.5 mL, 26 mmol, 8.4 equivalences) was slowly added while stirring at 0 °C for 30 min. Then, a solution of decahydronaphthyl-1,8-dione (500 mg, 3.1 mmol, 1.0 equivalences) in dichloromethane (0.5 mL) was added dropwise. The ice bath was removed, and the reaction mixture was heated to 90 °C for 3 h. The reaction mixture was cooled on crushed ice, and the product was extracted with dichloromethane (3 × 75 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a thick orange liquid. The crude product was used in the next step without further purification.

[0050] 2) Preparation of NIR-920;

[0051] The synthesis route of NIR-920 is as follows:

[0052]

[0053] Synthesis and characterization of NIR-920: 2,3,3-trimethyl-1-propyl-3H-indole-1-iodide (329 mg, 1 mmol), a linker (178 mg, 0.45 mmol), and 2,6-di-tert-butyl-4-methylpyridine (417 μL, 3 mmol) were added to a flame-dried flask and dissolved in a mixture of n-butanol (n-C4H9OH) (0.165 M) and toluene (0.35 M). The mixture was subjected to a freeze-vacuum-thaw cycle three times, followed by heating to 100 °C and holding for 30 min. The crude product was directly analyzed by reversed-phase chromatography (100 g C). 18 The aq (45% to 80% acetonitrile / water system) was purified and then lyophilized to obtain NIR-920 as a green solid. The 1H NMR spectrum of NIR-920 is shown below. Figure 10 As shown. 1H NMR (400MHz, Methanol-d4) δ8.46–8.34(m,2H),7.56(d,J=7.5Hz,2H),7.47(t,J=7.5Hz, 2H),7.41–7.36(m,2H),7.35–7.29(m,2H),6.48–6.24(m,2H),4.26–4.13(m,4H),3.02–2. 90(m,2H),2.77–2.64(m,1H),2.59–2.44(m,2H),2.31–2.20(m,2H),1.93(q,J=7.6Hz,4H ),1.77(s,12H),1.56–1.46(m,2H),1.09(t,J=7.3Hz,6H).LC-HRMS(ESI-TOF):calcd.for C 40 H 47 Cl2N2 + [M] + 625.3111; found 625.3111.

[0054] 3) Preparation of NIR-940;

[0055] The synthesis route of NIR-940 is as follows:

[0056]

[0057] Synthesis and characterization of NIR-940: 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-bromide (354 mg, 1 mmol), a linker (178 mg, 0.45 mmol), and 2,6-di-tert-butyl-4-methylpyridine (417 μL, 3 mmol) were added to a flask dried in a high-temperature oven and dissolved in a mixture of n-butanol (n-C4H9OH) (0.165 mol / L) and toluene (0.35 mol / L). The mixture was subjected to a freeze-vacuum-thaw cycle three times, followed by heating to 100 °C and holding for 30 min. The crude product was directly analyzed by reversed-phase chromatography (100 g C... 18 The aq (30% to 70% acetonitrile / water system) was purified and then lyophilized to give NIR-940 as a dark green solid. The 1H NMR spectrum of NIR-940 is shown below. Figure 11 As shown. 1H NMR (400MHz, Methanol-d4) δ8.39(d,J=14.1Hz,2H),7.55(d,J=7.4Hz,2H),7.46(t,J=7.7Hz,2H),7.3 7(d,J=8.0Hz,2H),7.32(t,J=7.4Hz,2H),6.36(d,J=14.2Hz,2H),4.22(t,J=7.5Hz,4H),3.80–3.73(m ,2H),3.00–2.92(m,2H),2.74–2.65(m,1H),2.56–2.47(m,2H),2.32–2.28(m,4H),2.26–2.20(m,2H), 1.93–1.87(m,4H),1.76(s,12H),1.73–1.70(m,4H),1.55–1.50(m,4H).LC-HRMS(ESI-TOF):calcd.for C 46 H 55 Cl2N2O4 + [M] + 769.3533; found 769.3536.

[0058] In the above synthesis process, the starting material decahydronaphthalene-1,8-dione undergoes oxidation and substitution reactions with phosphorus oxychloride and dichloromethane (as indicated by the arrows above the route), thus yielding a conjugated linker. 2,3,3-trimethyl-1-propyl-3H-indole-1-iodide and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-bromide molecules react with the obtained linker under weakly base catalysis in a mixed solvent of n-butanol and toluene, respectively, to yield NIR-920 and NIR-940.

[0059] The above scheme is a preferred embodiment. In the preparation process, any one or a combination of several of methanol, ethanol, acetonitrile, n-butanol, toluene, xylene, acetic acid or acetic anhydride can be selected as the reaction solvent; any one or a combination of several of triethylamine, N,N-diisopropylethylamine, 2,6-di-tert-butyl-4-methylpyridine, ammonium acetate, potassium acetate, sodium acetate can be selected as the weak base catalyst; and a certain temperature from room temperature to the reflux temperature of the solvent can be selected as the reaction temperature, including but not limited to the above combinations.

[0060] As a preferred embodiment of the present invention, a nonamethyst dye with the following structure is synthesized:

[0061]

[0062] NIR-940 was selected for further investigation;

[0063] For NIR-940 or nonamethyne molecular analog dyes conforming to the following general structural formula I:

[0064] General Formula I:

[0065] NIR-940 or a nonamethyne dye molecule conforming to general formula I can covalently bind to serum albumin in situ in vivo; NIR-940 or a nonamethyne dye molecule conforming to general formula I can also be incubated with human serum albumin (HSA) in vitro under heating conditions to obtain a covalent complex of the two, NIR-940@HSA.

[0066] The preparation process of the intravenous injection working solutions of NIR-940 and NIR-940@HSA is as follows:

[0067] Preparation of NIR-940 working solution:

[0068] Step a. Dissolve NIR-940 in DMSO to form a NIR-940 stock solution with a concentration of 20 mmol / L;

[0069] Step b. Dissolve 5 μL of NIR-940 stock solution directly in 200 μL of PBS to obtain a working solution of NIR-940 with a concentration of 500 μmol / L;

[0070] Preparation of NIR-940@HSA working solution:

[0071] Step c. Dissolve human serum albumin solid in PBS to obtain a protein solution with a concentration of 10 μmol / L;

[0072] Step d. Add 5 μL of NIR-940 stock solution to 10 mL of protein solution, mix thoroughly by shaking, and then incubate in a 50°C oven for 2 h. During this process, the molar ratio of nonamethystine dye NIR-940 to human serum albumin is 1:1. It should be noted that the incubation temperature, duration, heating method, and the concentration ratio of dye to protein can all be adjusted according to experimental requirements; the final product will always be a dye-protein complex.

[0073] Step e. Concentrate the mixed solution obtained in step d to 200 μL using an ultrafiltration centrifuge tube at a speed of 8000-10000 rpm to obtain a NIR-940@HSA working solution with a concentration of 500 μmol / L.

[0074] Through the above steps, NIR-940 working solution and NIR-940@HSA working solution were successfully prepared. The HSA protein in NIR-940@HSA specifically refers to human serum albumin, but may also include NIR-940@Protein, a dye-protein complex formed by NIR-940 and its structural analogs with other different proteins. These proteins include, but are not limited to, various albumins such as human serum albumin (HSA), bovine serum albumin (BSA), mouse serum albumin (MSA), and chicken ovalbumin (OVA), as well as β-lactoglobulin and other proteins.

[0075] like Figure 1 As shown in Figure 1, the absorption and fluorescence emission spectra of NIR-940 in dimethyl sulfoxide, mouse serum, and phosphate buffered aqueous solution are presented respectively. The fluorescence intensity of NIR-940 under 915 nm excitation in different solutions is compared. The results show that NIR-940 is in an aggregation-induced quenched state in phosphate buffered aqueous solution (the main absorption peak at 747 nm is significantly blue-shifted compared to 943 nm in dimethyl sulfoxide), while it exhibits significant fluorescence enhancement in mouse serum (the main absorption peak at 928 nm is significantly red-shifted compared to phosphate buffered aqueous solution, and not much different from that in dimethyl sulfoxide), reaching 86.6% of the fluorescence intensity in the organic solvent dimethyl sulfoxide.

[0076] like Figure 2 As shown, gel electrophoresis results of mouse serum collected at different time points after intravenous injection of NIR-940 at 500 μmol / L showed ( Figure 2 In section a), the band at 60-75 kDa represents the fluorescent band of mouse albumin in serum bound to NIR-940. The fluorescent band of the free dye below is almost invisible, indicating near-complete binding, demonstrating that NIR-940 can efficiently covalently bind to endogenous mouse serum albumin in vivo. Gel electrophoresis results after incubating 30 μmol / L NIR-940 with different concentrations of mouse serum (dye-to-protein ratio from 1:1 to 1:25) at 37°C for 2 hours in vitro showed that… Figure 2 In (b), even when the extracted mouse serum was diluted to 30 μmol, it was still able to bind rapidly to NIR-940, with only a small amount of free dye observed, demonstrating its effectiveness against [various irradiations / contaminations]. Figure 2 The statistical diagram of the fluorescence intensity of the free dye bands on the gel electrophoresis bands obtained in b is shown. Figure 2 c) When the serum concentration was increased to 150 micromoles (the dye to protein concentration ratio was 1:5), no free dye could be observed at all, indicating that complete binding was possible under this concentration condition.

[0077] like Figure 3As shown, the gel electrophoresis results after incubating 10 μmol / L NIR-940 with 10 μmol / L human serum albumin at different temperatures for 2 hours in vitro showed that ( Figure 3 In section a), NIR-940 exhibits a high binding efficiency to exogenous human serum albumin. Even after incubation at 37°C or even room temperature, only a small amount of free dye is observed. When the incubation temperature is greater than or equal to 50°C, no free dye is observed at all. Gel electrophoresis results after incubating 5-50 μmol / L NIR-940 with 10 μmol / L human serum albumin (protein to dye concentrations ranging from 1:0.5 to 1:5) at 50°C for 2 hours in vitro showed that… Figure 3 In section b), the fluorescence intensity of the fluorescent protein band increased with increasing dye concentration, reaching a peak at a ratio of 1:5. This suggests that human serum albumin may bind multiple dyes. Gel electrophoresis results of incubating 10 μmol / L NIR-940 and 10 μmol / L human serum albumin at 50°C for different times in vitro showed that… Figure 3 In step c), the intensity of the fluorescence band increased with prolonged incubation time, reaching a peak at 1-2 hours. After 0.5 hours, no free dye was observed, indicating that NIR-940 had almost completely covalently bound to HSA. Subsequent heating further adjusted its conformation to achieve optimal fluorescence emission. Meanwhile, for… Figure 3 Statistical results of fluorescence intensity of fluorescent protein bands and free dye bands under different incubation conditions in AC also support the above conclusion. Figure 3 (d).

[0078] like Figure 4 As shown, the high-resolution mass spectrometry results of human serum albumin (HSA) are... Figure 4 (a) and the high-resolution mass spectrometry results of NIR-940@HSA obtained after incubating 10 μmol / L NIR-940 and 10 μmol / L human serum albumin in vitro at 50°C for 2 h. Figure 4 b) together show that after NIR-940 and HSA are combined under the preferred incubation conditions, the molecular weight of HSA is completely unobservable, and only the molecular weight of NIR-940@HSA is observed, which proves that the two are completely combined.

[0079] like Figure 5 As shown, the above experiments demonstrate that the process of NIR-940 covalently binding with exogenous human serum albumin in vitro to obtain NIR-940@HSA, as well as the process of NIR-940 covalently binding with endogenous mouse albumin in serum after direct intravenous injection, are both rapid, efficient, and complete, and are suitable for subsequent tumor imaging or targeted imaging of the brain of stroke-affected mice.

[0080] like Figure 6As shown, the results of targeted imaging of the brains of stroke mice of different severities at different time points after direct tail vein injection of NIR-940 showed that ( Figure 6 In (a), the brains of stroke patients of varying severity could be well monitored over a longer time window and were well distinguished from the undamaged control group. Figure 6 The changes in fluorescence intensity over time in stroke regions of different severity in region a indicate that ( Figure 6 In the case of stroke (b), the fluorescence intensity generally increases with increasing severity. Under any severity, the fluorescence intensity in the stroke area first increases and then decreases over time. Figure 6 The change in the ratio of fluorescence signal to background signal over time in stroke regions of different severities in region a indicates that ( Figure 6 In group c), except for the very severe damage group, the signal-to-noise ratio (SNR) of all other groups increased with the severity of the damage. However, the SNR of the very severe damage group decreased, which is likely due to the significant increase in background signal caused by the greater degree of damage. Figure 6 The changes in fluorescence-labeled lesion area over time in stroke regions of different severities in region a indicate that ( Figure 6 (d) As the severity of the injury increases, the area of ​​damage shows a regular upward trend. These results strongly demonstrate that NIR-940 has excellent targeted imaging and diagnostic effects on the brain of stroke patients in mice.

[0081] like Figure 7 As shown, whole-body images of tumor-bearing mice in supine and lateral positions at different time points after NIR-940 was injected via the tail vein for subcutaneous xenografting of 4T1 tumors. Figure 7 (a) and whole-body imaging at different time points in supine and lateral positions of tumor-bearing mice that underwent subcutaneous xenografting of 4T1 tumors after NIR-940@HSA was injected via the tail vein. Figure 7 Both (b) and (c) demonstrate that both NIR-940 and NIR-940@HSA are capable of high-contrast imaging monitoring of tumor regions. Figure 7 Changes in tumor, liver, and skin signaling over time in mice in group b ( Figure 7 c), and Figure 7 Changes in tumor, liver, and skin signaling over time in mice in group a (a) Figure 7 (d) Together, it shows that in the NIR-940@HSA group, the liver and tumor signals first increased to 12 hours and then decreased, with a generally consistent trend. However, in the NIR-940 group, the liver signal generally showed a decreasing trend, while the tumor signal first increased to about 12 hours before decreasing. The changes in the tumor-liver signal ratio and tumor-skin signal ratio over time after injection of NIR-940 and NIR-940@HSA, respectively, indicate ( Figure 7In section e), the peak signal-to-noise ratio (SNR) of the tumor region in the NIR-940 group was slightly higher than that in the NIR-940@HSA group. During the time intervals when the SNR of the tumor skin in the NIR-940 group was higher than that in the NIR-940@HSA group, the tumor liver signal ratio was also higher in the NIR-940 group. These results indicate that both NIR-940 and NIR-940@HSA have good targeting effects on 4T1 tumors. However, due to the completely different trends in liver signal changes after injection, NIR-940 exhibits a lower tumor liver signal ratio and better tumor-targeting imaging effect 6 hours after injection.

[0082] like Figure 8 As shown, NIR-940, administered intravenously to mice with skin lesions on their backs, enabled long-term monitoring of the lesion area. Figure 8 In section a), both the signal-to-noise ratio and the leakage area first increase and then decrease over time. Figure 8 (b and c) represent NIR-940 binding to endogenous albumin in vivo, initially leaking and accumulating in the dorsal injury area, and then gradually being metabolized from the injury area. Similarly, NIR-940, administered intravenously to mice with scalp injuries, allows for long-term monitoring of the scalp injury area. Figure 8 In the middle (d), both the signal-to-noise ratio and the leakage area first increase and then decrease over time. Figure 8 In Figures e and f), NIR-940 binds to endogenous albumin in vivo, initially leaking and accumulating gradually in the scalp injury area, and then rapidly metabolizing from the injury area. These results demonstrate the excellent targeting ability of NIR-940 for skin injuries and its high imaging signal-to-noise ratio.

[0083] like Figure 9 As shown, NIR-940 was intravenously injected into mice in the blood-testis barrier disruption group and the control group, respectively. Figure 9 (a) The results showed that the testicular region of mice in the blood-testis barrier disruption group was well illuminated, while the testicular region of mice in the control group was not significantly illuminated. The signal-to-noise ratio and fluorescence intensity of the testicular region in the blood-testis barrier disruption group were significantly higher than those in the control group at most monitoring time points. Figure 9 (b and c). Additionally, NIR-940 was intravenously injected into mice in the intestinal vascular barrier disruption group and the control group, respectively. Figure 9(d) The results showed that since NIR-940 is mainly excreted through hepatobiliary metabolism and fecal metabolism after intravenous injection, significant intestinal signals were observed in both the control group and the intestinal vascular barrier disruption group before 24 hours of imaging. At the 24-hour and 48-hour imaging time points, the intestinal signal in the control group decreased significantly because most of the NIR-940 was metabolized through feces, while the signal in the intestinal lesion area of ​​the intestinal vascular barrier disruption group remained intact. At 24-hour and 48-hour imaging signal-to-noise ratio and fluorescence signal intensity in the lesion area, the intestinal vascular barrier disruption group mice were significantly higher than the control group mice. Figure 9 (e and f). The above results demonstrate that NIR-940 can accurately target and image the damaged areas of the blood-testis barrier and the intestinal vascular barrier with high contrast, and shows significant differences compared to the control group.

[0084] 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 nonamethyst dye probe, characterized in that, It is a nonamethyst dye, NIR-940, with the following structure: ； NIR-940 covalently binds to serum albumin in situ in vivo; NIR-940 is incubated with human serum albumin in vitro under heating conditions to obtain the covalent complex NIR-940@HSA.

2. The nonamethyst dye probe according to claim 1, characterized in that, The specific preparation process of the working solution of NIR-940@HSA is as follows: Step a. Dissolve NIR-940 in DMSO to form a NIR-940 stock solution with a concentration of 20 mmol / L; Step b. Dissolve 5 μL of NIR-940 stock solution directly in 200 μL of PBS to obtain a working solution of NIR-940 with a concentration of 500 μmol / L; Step c. Dissolve human serum albumin solid in PBS to obtain a protein solution with a concentration of 10 μmol / L; Step d. Add 5 μL of NIR-940 stock solution to 10 mL of protein solution, mix thoroughly by shaking, and then incubate in an oven at 50 °C for 2 h; Step e. Concentrate the mixed solution obtained in step d to 200 μL using an ultrafiltration centrifuge tube at a speed of 8000-10000 rpm to obtain a NIR-940@HSA working solution with a concentration of 500 μmol / L.

3. The nonamethyst dye probe according to claim 2, characterized in that, In the NIR-940@HSA working solution, the molar ratio of NIR-940 to human serum albumin is 1:

1.

4. The nonamethyst dye probe according to claim 1, characterized in that, The preparation steps of the NIR-940 are as follows: Preparation of the linker: A mixture of N,N-dimethylformamide and dichloromethane was added to a flask dried in a high-temperature oven and equipped with a magnetic stir bar; the flask was purged with argon and cooled to 0°C; phosphorus oxychloride was slowly added while stirring at 0°C for 30 min; then decahydronaphthalene-1,8-dione was added dropwise; the ice bath was removed, and the reaction mixture was heated to 90°C and reacted for 3 h; the reaction mixture was poured onto crushed ice to cool, and the product was extracted with dichloromethane; the combined organic layers were dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a thick orange liquid; Preparation of NIR-940: 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-bromide, a linker, and 2,6-di-tert-butyl-4-methylpyridine were added to a flask dried in a high-temperature oven and dissolved in a mixture of n-butanol and toluene. The mixture was subjected to a freeze-vacuum-thaw cycle three times, and then heated to 100°C and held for 30 min. The crude product was directly purified by reversed-phase chromatography and then lyophilized to obtain NIR-940.

5. The application of a nonamethystine dye probe according to any one of claims 1-4 in the preparation of tumor-targeted imaging diagnostic reagents, characterized in that, In vascular imaging, reagents are injected directly into the vein for imaging.

6. The application of a nonamethystine dye probe according to any one of claims 1-4 in the preparation of a targeted imaging diagnostic reagent for the brain of stroke-affected mice, characterized in that, The reagents are used directly for imaging via tail vein injection.

7. The application of a nonamethystine dye probe according to any one of claims 1-4 in the preparation of targeted imaging diagnostic reagents for skin lesions, characterized in that, The reagents are used directly for imaging via tail vein injection.

8. The application of a nonamethystine dye probe according to any one of claims 1-4 in the preparation of a targeted imaging diagnostic reagent for blood-testis barrier disruption, characterized in that, The reagents are used directly for imaging via tail vein injection.

9. The application of a nonamethystine dye probe according to any one of claims 1-4 in the preparation of a targeted imaging diagnostic reagent for intestinal vascular barrier disruption, characterized in that, The reagents are used directly for imaging via tail vein injection.

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