Near-infrared two-region albumin efficient in-situ targeting cyanine dye as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of near-infrared imaging probes, and provides a near-infrared two-region albumin efficient in-situ targeting cyanine dye as well as a preparation method and application thereof. The structural formula of the cyanine dye is # imgabs 0 #, R structure is I or II: # imgabs 1 #, and n is any one of 2-10. The cyanine dye provided by the invention can quickly form stable covalent binding with albumin in situ in an in-vitro or in-vivo physiological environment so as to generate bionic fluorescent protein with bright luminescence and high biocompatibility. The in-situ bionic fluorescent protein construction strategy provided by the invention not only can realize high-resolution mouse angiography, but also can realize real-time and accurate monitoring on blood-brain barrier damage accompanying cerebral ischemic stroke of the mouse. The dye provided by the invention can realize multi-channel joint monitoring with angiographic contrast agents with different excitation wavelengths, and realizes simultaneous marking of cerebral vascular and brain injury after cerebral apoplexy.

Description

Technical Field

[0001] The present invention belongs to the technical field of near-infrared imaging probes, and particularly relates to a cyanine dye with high-efficiency in-situ targeting of albumin in the second near-infrared region, and a preparation method and application thereof. Background Art

[0002] The blood-brain barrier is a highly selective semi-permeable interface between the blood and the brain, which is responsible for regulating the exchange of nutrients, metabolites, and other substances between the blood and the brain parenchyma, and protecting the central nervous system from potential neurotoxic substances in the blood. The dysfunction and disruption of the blood-brain barrier have attracted extensive attention as a common feature of many common neurological diseases (such as stroke, traumatic brain injury, hypertension, and epilepsy, etc.). Therefore, achieving high-sensitivity real-time in-vivo visualization of blood-brain barrier disruption is of great significance for studying the pathogenesis of these diseases and evaluating the effectiveness of therapeutic interventions. Currently, in clinical diagnosis, imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging and computed tomography are commonly used to measure the changes in vascular permeability and the occurrence of blood-brain barrier dysfunction. However, these methods have some limitations, including: 1) a long scanning time is required to obtain clear imaging; 2) the biological safety is reduced due to ionizing radiation and contrast agent residues; 3) the imaging contrast is poor. Therefore, it is particularly urgent to develop an imaging technique that is safe, highly sensitive, and can accurately locate the damaged area of the blood-brain barrier.

[0003] Near-infrared second region (NIR-II, 1000 - 1700 nm) imaging technology has higher imaging contrast and tissue penetration depth compared with visible light and near-infrared first region (NIR-I, 700 - 900 nm) imaging technology, and has been used in various disease imaging and surgical navigation. NIR-II fluorescent dyes have also been widely developed. In order to achieve a high-sensitivity targeting effect, targeting peptides or antibodies are usually added, but these exogenous modifications often face challenges such as immune system uptake and low-specificity labeling. In recent years, a new strategy that combines the high selectivity of protein tags and the bright and wavelength-tunable advantages of synthetic organic fluorophores has emerged, that is, using organic small-molecule fluorophores to be anchored on gene-encoded protein tags through covalent or non-covalent interactions for bioimaging. This bionic fluorescent protein can be formed under physiological conditions in vivo through a simple incubation reaction without a complex genetic encoding process, achieving high-contrast bioimaging.

[0004] The change in blood-brain barrier permeability is usually visually evaluated by detecting the extravasation amount of an exogenous tracer in brain tissue after vascular infusion. Currently, Evans blue (EB) and clinically available indocyanine green (ICG) have been used for the evaluation of blood-brain barrier damage based on their potential serum albumin-binding ability. Although the addition of dyes and tracers has made great contributions to the study of blood-brain barrier disruption, there are still challenges: 1) The binding specificity of dyes to albumin is limited, often resulting in random accumulation in other tissues and organs of the organism, which may cause serious adverse reactions; 2) The detection methods are mostly naked-eye observation or NIR-I imaging after tissue ex vivo, lacking high-resolution, real-time non-invasive in vivo imaging means. Therefore, the development of NIR-II contrast agents suitable for high-resolution, real-time in vivo visualization of blood-brain barrier damage has important basic research and clinical significance. For this reason, the present invention proposes a cyanine dye with efficient in-situ targeting of albumin in the second near-infrared region, its preparation method and application. Summary of the Invention

[0005] The purpose of the present invention is to provide a cyanine dye with efficient in-situ targeting of albumin in the second near-infrared region, its preparation method and application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A cyanine dye with efficient in-situ targeting of albumin in the second near-infrared region, the structural formula of the cyanine dye is:

[0008]

[0009] Among them, the R structure is I or II:

[0010]

[0011] Among them, n is any value from 2 to 10.

[0012] Furthermore, when the R structure is I, the dye is Cx-808, x = n + 2; when the R structure is II, the dye is Cx-1080, x = n + 2.

[0013] According to the preparation method of the cyanine dye described above, the specific preparation steps are as follows:

[0014] When the R structure is I and n = 2, a mixture of compound a and ethyl 4-bromobutyrate is dissolved in acetonitrile and heated under reflux overnight to obtain compound b; compound b is added to an aqueous hydrobromic acid solution and heated under reflux for 4 h to form compound c; compound c, compound d and sodium acetate anhydrous are dissolved in ethanol and stirred at 50 °C overnight to form cyanine dye C4-808; wherein, the structural formulas of compound a, compound b, compound c and compound d are as follows:

[0015]

[0016] When the R structure is I and n = 3 - 10, compound a and compound e are mixed and heated under reflux overnight to obtain compound c; compound c, compound d and sodium acetate anhydrous are dissolved in ethanol and stirred at 80 °C overnight to form cyanine dye Cx-808, where x = 5 - 12; wherein, the structural formula of compound e is as follows:

[0017]

[0018] When the R structure is II, compound f and compound i react in N,N-dimethylformamide catalyzed by potassium carbonate to form compound g; compound g is mixed with HCl and reacted at 100 °C to form compound h; compound h, compound d and 2,6-di-tert-butyl-4-methylpyridine are dissolved in a mixed solution of toluene and n-butanol and reacted at 100 °C to form cyanine dye Cx-1080; wherein, the structural formulas of compound i, compound f, compound g and compound h are as follows:

[0019]

[0020] Application of the cyanine dye prepared by the preparation method of the cyanine dye described above in the preparation of a biomimetic fluorescent protein.

[0021] Preparation method of a biomimetic fluorescent protein, the biomimetic fluorescent protein is a stable covalent complex formed by the cyanine dye prepared by the preparation method of the cyanine dye described above and serum albumin. Based on the strong affinity between the cyanine dye and serum albumin, the cyanine dye can rapidly form a stable covalent bond with albumin in vitro or in vivo physiological environment in situ to generate a biomimetic fluorescent protein with bright luminescence and high biocompatibility. The specific preparation method is as follows:

[0022] Method 1: Human serum albumin is dissolved in a phosphate buffer solution to form an albumin solution, and 10 μM cyanine dye Cx-1080 is added; the mixed system is placed in a light-shielded thermostatic shaker and incubated at room temperature to 60 °C for 0 - 24 h; after incubation, the mixture is separated by 12% SDS-PAGE electrophoresis;

[0023] Method 2: Inject 200 μL of 600 μM cyanine dye Cx-1080 into a mouse via the tail vein. In the physiological environment, the cyanine dye and serum albumin in situ form a biomimetic fluorescent protein.

[0024] Furthermore, in Method 1 and Method 2, x in the cyanine dye Cx-1080 both takes the value of 7, that is, n = 5; in Method 1, the optimal incubation temperature is 37 - 60 °C, and the optimal incubation time is 0 - 12 h;

[0025] The binding rate and binding efficiency of the dye to albumin are related to the albumin concentration. The higher the albumin concentration, the faster the binding rate of the dye to albumin and the higher the binding efficiency;

[0026] The optical properties of the dye are related to the side chain length. When n = 5, the dye molecule has the best optical properties; when n < 5, the optical properties of the molecule deteriorate as the chain length decreases; when n > 5, the optical properties of the molecule deteriorate as the chain length increases;

[0027] The binding rate and binding efficiency of the dye to albumin are related to the side chain length. When n = 5, the dye molecule has the fastest binding rate and the highest binding efficiency with albumin; when n < 5, the binding rate and binding efficiency of the dye molecule to albumin deteriorate as the chain length decreases; when n > 5, the binding rate and binding efficiency of the dye molecule to albumin deteriorate as the chain length increases.

[0028] The cyanine dye can quickly bind to serum albumin in the organism to form a bright and stable biomimetic fluorescent protein with a dye-protein structure, having good bioimaging ability and a longer metabolic half-life. Therefore, the biomimetic fluorescent protein prepared according to the above-mentioned preparation method of the biomimetic fluorescent protein can be applied to the preparation of vascular imaging reagents, including high-resolution imaging of the whole body blood vessels and local blood vessels of mice.

[0029] The cyanine dye can quickly and specifically bind to albumin leaked at the tissue damage site of the organism to in situ form a bright and stable biomimetic fluorescent protein, having the ability to label the damage location and evaluate the damage degree. Therefore, the biomimetic fluorescent protein prepared according to the above-mentioned preparation method of the biomimetic fluorescent protein can be applied to the preparation of tissue barrier breakage monitoring reagents, including but not limited to tissue damage localization and damage degree evaluation such as blood-brain barrier (BBB) damage and skin-blood vessel barrier damage.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1. The cyanine dye provided by the present invention regulates the luminescence performance of the dye and its binding efficiency with albumin by changing the length of the molecular side chain, thereby realizing rapid and efficient preparation of biomimetic fluorescent proteins in vitro and in situ in vivo.

[0032] 2. The cyanine dye provided by the present invention has the best luminescence performance compared with similar dyes, and the bionic fluorescent protein formed after combining with albumin has excellent biological imaging ability and photostability, which effectively solves the problems of aggregation fluorescence quenching and poor stability of near-infrared contrast agents.

[0033] 3. The in situ biomimetic fluorescent protein construction strategy provided by the present invention can realize real-time monitoring of blood-brain barrier damage and assessment of the degree of damage after mouse stroke, providing a richer theoretical basis for clinical research.

[0034] 4. The cyanine dye provided by the present invention can realize multi-channel joint monitoring with vascular contrast agents with different excitation wavelengths, thereby achieving simultaneous labeling of cerebral blood vessels and brain damage after stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The synthetic route of the cyanine dye Cx-1080 in Example 1; wherein a is the synthetic route of the cyanine dye C4-808; b is the synthetic route of the cyanine dye Cx-808 (x=5-12); and c is the synthetic route of the cyanine dye Cx-1080.

[0036] Figure 2 For compound C4-1080 1 H-NMR spectrum.

[0037] Figure 3 For compound C5-1080 1 H-NMR spectrum.

[0038] Figure 4 For compound C6-1080 1 H-NMR spectrum.

[0039] Figure 5 For compound C7-1080 1 H-NMR spectrum.

[0040] Figure 6 For compound C8-1080 1 H-NMR spectrum.

[0041] Figure 7 For compound C9-1080 1 H-NMR spectrum.

[0042] Figure 8 For compound C10-10801 1H-NMR spectrum.

[0043] Figure 9 of compound C11-1080 1 1H-NMR spectrum.

[0044] Figure 10 of compound C12-1080 1 1H-NMR spectrum.

[0045] Figure 11 of compound C4-1080 13 13C-NMR spectrum.

[0046] Figure 12 of compound C5-1080 13 13C-NMR spectrum.

[0047] Figure 13 of compound C6-1080 13 13C-NMR spectrum.

[0048] Figure 14 of compound C7-1080 13 13C-NMR spectrum.

[0049] Figure 15 of compound C8-1080 13 13C-NMR spectrum.

[0050] Figure 16 of compound C9-1080 13 13C-NMR spectrum.

[0051] Figure 17 of compound C10-1080 13 13C-NMR spectrum.

[0052] Figure 18 of compound C11-1080 13 13C-NMR spectrum.

[0053] Figure 19 High-resolution mass spectrum of compound C4-1080.

[0054] Figure 20 High-resolution mass spectrum of compound C5-1080.

[0055] Figure 21 High-resolution mass spectrum of compound C6-1080.

[0056] Figure 22 High-resolution mass spectrum of compound C7-1080.

[0057] Figure 23 High-resolution mass spectrum of compound C8-1080.

[0058] Figure 24 High-resolution mass spectrum of compound C9-1080.

[0059] Figure 25 High-resolution mass spectrum of compound C10-1080.

[0060] Figure 26 High-resolution mass spectrum of compound C11-1080.

[0061] Figure 27 High-resolution mass spectrum of compound C12-1080.

[0062] Figure 28 For compound C4-808 1 1H-NMR spectrum.

[0063] Figure 29 For compound C5-808 1 1H-NMR spectrum.

[0064] Figure 30 For compound C6-808 1 1H-NMR spectrum.

[0065] Figure 31 For compound C7-808 1 1H-NMR spectrum.

[0066] Figure 32 For compound C8-808 1 1H-NMR spectrum.

[0067] Figure 33 For compound C9-808 1 1H-NMR spectrum.

[0068] Figure 34 For compound C10-808 1 1H-NMR spectrum.

[0069] Figure 35 For compound C11-808 1 1H-NMR spectrum.

[0070] Figure 36 For compound C12-808 1 1H-NMR spectrum.

[0071] Figure 37Basic optical properties of dye Cx-1080; where a is the fluorescence intensity of the dye in chloroform, dimethyl sulfoxide, methanol, and water; b is the absorption spectrum of the dye in dimethyl sulfoxide; c is the emission spectrum of the dye in dimethyl sulfoxide; d is the photostability of the dye in dimethyl sulfoxide.

[0072] Figure 38 Fluorescence intensities of dye Cx-1080 and human serum albumin after incubation at different reaction temperatures and reaction times in Example 2; where a is incubation for 0 minutes at room temperature; b is incubation for 0 minutes at 37 °C; c is incubation for 0 minutes at 60 °C; d is incubation for 2 h at room temperature; e is incubation for 2 h at 37 °C; f is incubation for 2 h at 60 °C; g is incubation for 12 h at room temperature; h is incubation for 12 h at 37 °C; i is incubation for 12 h at 60 °C; j is incubation for 24 h at room temperature; k is incubation for 24 h at 37 °C; l is incubation for 24 h at 60 °C.

[0073] Figure 39 SDS-PAGE electrophoresis separation results of dye Cx-1080 and human serum albumin after incubation at different reaction temperatures and reaction times in Example 2; where a is incubation for 0 minutes at different temperatures; b is incubation for 2 h at different temperatures; c is incubation for 12 h at different temperatures; d is incubation for 24 h at different temperatures.

[0074] Figure 40 Fluorescence intensities of dye Cx-1080 and human serum albumin after incubation at different temperatures and different protein concentrations in Example 3; where a is incubation for 0 minutes at room temperature (10 μM HSA); b is incubation for 0 minutes at room temperature (1% HSA); c is incubation for 0 minutes at room temperature (5% HSA); d is incubation for 2 h at room temperature (10 μM HSA); e is incubation for 2 h at room temperature (1% HSA); f is incubation for 2 h at room temperature (5% HSA); g is incubation for 2 h at 37 °C (10 μM HSA); h is incubation for 2 h at 37 °C (1% HSA); i is incubation for 2 h at 37 °C (5% HSA); j is incubation for 2 h at 60 °C (10 μM HSA); k is incubation for 2 h at 60 °C (1% HSA); l is incubation for 2 h at 60 °C (5% HSA).

[0075] Figure 41 SDS-PAGE electrophoresis separation results of dye Cx-1080 and human serum albumin after incubation at different reaction temperatures and different protein concentrations in Example 3; where a is incubation for 0 minutes at different protein concentrations and room temperature; b is incubation for 2 h at different protein concentrations and room temperature; c is incubation for 2 h at different protein concentrations and 37 °C; d is incubation for 2 h at different protein concentrations and 60 °C.

[0076] Figure 42 Statistical results of ion mobility mass spectrometry in Example 3.

[0077] Figure 43 For the different domains of the dye and human serum albumin in Example 4.

[0078] Figure 44 For the results of proteomic analysis after the binding of the dye and human serum albumin in Example 4.

[0079] Figure 45 For the exploration of the ability of the cyanine dye and serum albumin to form a biomimetic fluorescent protein in vivo in Example 5; where a is the angiography of the hind limb blood vessels of the mouse and the statistical results of the imaging signal-to-noise ratio after intravenous injection of the dye; b is the fluorescence intensity of the mouse blood and the results of SDS-PAGE electrophoresis separation after intravenous injection of the dye; c is the fluorescence intensity of the mouse serum and the results of SDS-PAGE electrophoresis separation after intravenous injection of the dye.

[0080] Figure 46 For the exploration of the real-time monitoring and the monitoring of the damage degree of the blood-brain barrier injury accompanied by stroke by the biomimetic fluorescent protein in Example 6; a is the fluorescence imaging map for monitoring the blood-brain barrier injury accompanied by photochemical cerebral thrombosis in mice by constructing an in-situ biomimetic fluorescent protein; b is the statistical results of the imaging fluorescence signals for monitoring the blood-brain barrier injury accompanied by photochemical cerebral thrombosis in mice by constructing an in-situ biomimetic fluorescent protein; c is the fluorescence imaging map of the excised brain tissue of the mouse after photochemical cerebral thrombosis modeling and injection of the dye by constructing an in-situ biomimetic fluorescent protein; d is the statistical results of the imaging fluorescence signals of the excised brain tissue of the mouse after photochemical cerebral thrombosis modeling and injection of the dye by constructing an in-situ biomimetic fluorescent protein.

[0081] Figure 47 For the fluorescence imaging map for monitoring the degree of blood-brain barrier injury accompanied by photochemical cerebral thrombosis in mice by constructing an in-situ biomimetic fluorescent protein in Example 6.

[0082] Figure 48 For the dual-color imaging map of the blood-brain barrier injury and cerebral blood vessels accompanied by photochemical cerebral thrombosis in mice in Example 7. Detailed implementation manners

[0083] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0084] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0085] Example 1: This example provides a preparation method of a cyanine dye with high-efficiency in-situ targeting of albumin in the second near-infrared region.

[0086] The synthesis route of the cyanine dye C4-808 is as Figure 1 shown in a below, and the specific preparation steps are as follows:

[0087] (1) Compound a (10 mmol) and ethyl 4-bromobutyrate (29.8 mmol) were dissolved in 21.28 mL of acetonitrile and heated with stirring under reflux for 48 h; after the reaction system was cooled, the solvent was removed by a rotary evaporator, and the product was washed repeatedly with ether until it became a pink solid, obtaining compound j (compound j belongs to compound b, 7.5 mmol, 75%).

[0088] (2) Compound j (2 mmol) was dissolved in 3.45 mL of an aqueous solution of 30% hydrogen bromide and heated with stirring under reflux for 4 h. After the reaction was completed, it was cooled to room temperature and the solvent was removed by a rotary evaporator. White crystals precipitated by adding acetone to the remaining solid were compound k (compound k belongs to compound c, 0.72 mmol, 36%)

[0089] (3) Compound k (0.2 mmol), compound d (0.1 mmol) and sodium acetate (0.2 mmol) were dissolved in anhydrous ethanol (2 mL), and the reaction was carried out at 50 °C overnight. After cooling, the solvent of the system was removed and the product was separated by column chromatography to obtain a dark green product C4-808 (0.023 mmol, 23%).

[0090] The synthetic route of cyanine dye Cx-808 (x = 5 - 12) is as Figure 1 shown in b below, and the specific preparation steps are as follows:

[0091] (1) Compound a (10 mmol) and compound e (10 mmol) were added to a reaction vessel and heated to 110 °C for 12 h. After the reaction system was cooled to room temperature, acetone was added to wash the product, and this was repeated three times to obtain compound c (4.3 mmol, 43%).

[0092] (2) Compound c (0.2 mmol), compound d (0.1 mmol) and sodium acetate (0.2 mmol) were dissolved in anhydrous ethanol (2 mL), and the reaction was carried out at 80 °C overnight. After cooling, the solvent of the system was removed and the product was separated by column chromatography to obtain a dark green product Cx-808 (x = 5 - 12).

[0093] The synthetic route of cyanine dye Cx-1080 (x = 4 - 12) is as Figure 1 shown in c below, and the specific preparation steps are as follows:

[0094] (1) Based on compound f obtained by the method proposed in the invention patent with the application number CN202310217215.8, compound f (1.4 mmol), compound i with different chain lengths (1.6 mmol) and potassium carbonate (4.1 mmol) were dissolved in dry N,N-dimethylformamide (DMF), and in N 2Under protection, the reaction was stirred overnight at 80 °C. After the reaction was completed, saturated brine was added and the mixture was extracted with ethyl acetate multiple times to obtain the crude reaction product. After performing the water removal operation, the crude product was separated by column chromatography to obtain compound g (0.78 mmol, 56%).

[0095] (2) Compound g (0.46 mmol) was dissolved in concentrated hydrochloric acid (2 mL), and the reaction was heated at 100 °C for 2 h. After the reaction was completed, the solution was cooled to room temperature and 0.5 mL of potassium iodide (1 M) was added. The crude product was obtained by filtration, and the impurities were washed away with water and ethyl acetate to obtain compound h (0.33 mmol, 72%).

[0096] (3) Compound h (0.12 mmol), compound d (0.055 mmol), and 2,6-di-tert-butyl-4-methylpyridine (DTBMP, 0.37 mmol) were dissolved in a mixed solution of dry n-butanol (1.4 mL) and toluene (PhMe, 0.6 mL), and the reaction was carried out at 100 °C for 12 h. After the reaction was completed, the reaction mixture was filtered and purified in ethyl acetate to obtain the final product Cx-1080 (0.55 mmol, 45%).

[0097] The proton nuclear magnetic resonance ( 1 1H-NMR), carbon nuclear magnetic resonance ( 13 13C-NMR), and high-resolution mass spectrometry data of the cyanine dyes (Cx-1080, Cx-808) with efficient in situ targeting of NIR-II albumin prepared in the present invention are shown in Figures 2 - 36 .

[0098] Example 2: Cyanine dyes with different side chain lengths exhibited different optical properties.

[0099] As shown in Figure 37 a, dye Cx-1080 has absorption and emission peaks in the second near-infrared region in dimethyl sulfoxide (the fluorescence spectrum was measured under excitation by a 1064 nm wavelength laser), and among them, C7-1080 (n = 5) has the maximum absorption peak and fluorescence emission peak.

[0100] As shown in Figure 37 b, dye Cx-1080 showed different fluorescence intensities in dimethyl sulfoxide (DMSO), chloroform, methanol, and water, and among them, C7-1080 (n = 5) has the highest fluorescence intensity in any of the above solvents.

[0101] As shown in Figure 37As shown in FIGS. c-e, dye Cx-1080 has a similar fluorescence decay curve in dimethyl sulfoxide under continuous laser irradiation. The results show that the synthesized dye Cx-1080 of the present invention is less affected by photobleaching and has good photostability.

[0102] Example 3: Optimization of the conditions for the formation of a biomimetic fluorescent protein by a cyanine dye and human serum albumin in vitro and screening of the optimal cyanine dye.

[0103] The biomimetic fluorescent protein in this example is formed by a nucleophilic substitution reaction between the chlorine substituents in dye Cx-1080 and specific amino acid sites of human serum albumin to form a stable covalent bond. The method for constructing the biomimetic fluorescent protein in vitro includes:

[0104] Experiment 1: Dissolve human serum albumin (HSA) in a phosphate buffer solution to form an albumin solution with a protein concentration of 10 μM, and add dye Cx-1080 in a molar ratio of 1:1; place the mixed system in a light-shielded constant-temperature shaker and incubate at room temperature (RT), 37 °C, and 60 °C for 0 min, 2 h, 12 h, and 24 h respectively; and separate the incubated mixture by 12% SDS-PAGE electrophoresis.

[0105] The fluorescence intensity results are as Figure 38 shown. At a shorter incubation time, C7-1080 has the best fluorescence intensity; when incubating at room temperature, the fluorescence intensity of the dye increases with the extension of the incubation time. When the incubation time is longer than 12 h, the fluorescence intensity of the dye reaches a steady state (almost no longer changes); when incubating at 37 °C, the fluorescence intensity of the dye increases with the extension of the incubation time. When the incubation time is longer than 2 h, the fluorescence intensity of the dye reaches a steady state; when incubating at 60 °C, the fluorescence intensity of the dye increases with the extension of the incubation time. When the incubation time is longer than 2 h, the fluorescence intensity of the dye decreases with the extension of the incubation time.

[0106] The electrophoresis separation results are as Figure 39 shown. C7-1080 has the strongest albumin-binding ability under different incubation conditions; when incubating at room temperature, the free dye band decreases with the extension of the incubation time, and the dye-albumin complex band increases with the extension of the incubation time; when incubating at 37 °C, the free dye band decreases with the extension of the incubation time, and the dye-albumin complex band increases with the extension of the incubation time. When the incubation time is longer than 12 h, the binding of the dye to the protein reaches a steady state; when incubating at 60 °C, the free dye band decreases with the extension of the incubation time, and the dye-albumin complex band increases with the extension of the incubation time. When the incubation time is longer than 2 h, the protein structure is damaged and the fluorescence band disappears.

[0107] Experiment 2: Human serum albumin was dissolved in phosphate buffer solution to form albumin solutions with protein concentrations of 10 μM, 150 μM (1%, w / w), and 750 μM (5%, w / w), respectively. Dye Cx-1080 was added in a molar ratio of 1:1; the mixed system was incubated in a light-shielded constant-temperature shaker at room temperature, 37 °C, and 60 °C for 2 h respectively.

[0108] The fluorescence intensity results are as Figure 40 shown. Under different incubation conditions, C7-1080 has the best fluorescence intensity; in an environment with a low protein concentration (10 μM), the fluorescence intensity of the dye increases with the increase of the reaction temperature; in an environment with a medium protein concentration (150 μM), the fluorescence intensity of the dye increases with the increase of the reaction temperature. When the reaction temperature is higher than 37 °C, the fluorescence intensity of the dye reaches a steady state; in an environment with the concentration of in vivo serum protein (750 μM), the fluorescence intensity of the dye can quickly reach equilibrium at room temperature. When the reaction temperature is higher than room temperature, the fluorescence intensity of the dye reaches a steady state and does not change significantly.

[0109] The electrophoresis separation results are as Figure 41 shown. Under different incubation conditions, C7-1080 has the best albumin binding efficiency; in an environment with a low protein concentration (10 μM), the bands of the dye-albumin complex increase with the increase of the reaction temperature; in an environment with a medium protein concentration (150 μM), the fluorescence intensity of the dye increases with the increase of the reaction temperature. When the reaction temperature is higher than 37 °C, the binding of the dye to the protein reaches a steady state; in an environment with the concentration of in vivo serum protein (750 μM), the binding of the dye to albumin can quickly reach equilibrium at room temperature.

[0110] The above experiments prove that under in vitro conditions, C7-1080 has the strongest binding ability and binding speed with human serum albumin, and the reaction temperature is 37-60 °C and the reaction time is 0-12 h as the best reaction conditions; when the dye is in an environment with the concentration of biological serum albumin, it can quickly and efficiently bind to albumin to form a stable dye-protein complex, namely biomimetic fluorescent protein, at room temperature.

[0111] The ratio of protein to the formed biomimetic fluorescent protein in the above incubated system was quantitatively analyzed by ion mobility mass spectrometry. The results are as Figure 42 shown. Under the incubation conditions of room temperature, 37 °C, and 60 °C, C7-1080 all shows the best preparation efficiency of biomimetic fluorescent protein.

[0112] Example 4: Confirmation of the specific binding domain and binding site of the cyanine dye and human serum albumin to form biomimetic fluorescent protein.

[0113] Figure 43The high-resolution mass spectrometry detection results after incubation of the dye C7-1080 with different domains DI, DII, and DIII of human serum albumin are provided. It can be seen that domain DIII of human serum albumin has the strongest binding ability with the cyanine dye, demonstrating the binding of C7-1080 to domain DIII of human serum albumin.

[0114] As Figure 44 shown, the amino acid sites where C7-1080 binds to human serum albumin were analyzed by proteomics. It can be seen that the Cys477 site has the highest score, that is, the possibility of the Cys477 site reacting with C7-1080 to form a stable biomimetic fluorescent protein through a covalent bond is the greatest.

[0115] Example 5: Cyanine dyes and serum albumin have the ability to form biomimetic fluorescent proteins in vivo in whole blood in situ.

[0116] Figure 45 In a, the imaging data of the hind limb blood vessels of mice after tail vein injection of Cx-1080 (200 μL, 600 μM) are provided to observe the ability of the dye to form stable biomimetic fluorescent proteins with serum albumin in vivo in situ. It can be seen that C7-1080 has the best signal-to-noise ratio of blood vessel imaging and blood vessel fluorescence brightness, indicating that C7-1080 exhibits the best in situ biomimetic fluorescent protein preparation efficiency.

[0117] Figure 45 In b and c, the brightness of the blood and serum of mice and the SDS-PAGE electrophoresis separation results after tail vein injection of Cx-1080 (200 μL, 600 μM) are provided. It can be seen that C7-1080 exhibits the best blood / serum brightness, further indicating that C7-1080 has the best in situ biomimetic fluorescent protein preparation efficiency.

[0118] Example 6: The strategy of in situ constructing biomimetic fluorescent proteins with cyanine dyes and serum albumin can guide the real-time monitoring and damage degree monitoring of blood-brain barrier damage accompanied by stroke.

[0119] Establish a photochemical cerebral thrombosis animal model in mice: C57 mice (8-10 weeks old), with an average weight of 20 g. Isoflurane was used to anesthetize the mice during the operation. Bengal rose (0.4 mg, diluted with 0.2 mL PBS) was injected intravenously, and then the intact skull was irradiated with a 532 nm laser (diameter 2 mm, power density 1.59 W / cm 2 ) for 10 min. The laser was vertically centered, 1 mm behind the skull, and 2 mm outside the skull. At the same time, by adjusting the laser irradiation time, mild, moderate, and severe stroke injuries (Stroke) were simulated, which were 1 min, 5 min, and 10 min respectively; the sham operation group (Sham) did not undergo laser-induced surgery.

[0120] Two minutes after model establishment, C7-1080 dye was injected and excited with a 1064 nm laser, and imaging was performed at a wavelength of 1200 nm. As shown in Figure 46 a and b below, C7-1080 can perform real-time long-term monitoring of the disruption of the blood-brain barrier after brain injury, and there is sufficient imaging signal-to-noise ratio to distinguish between the injured side and the normal side; as shown in Figure 46 c and d below, the labeling of blood-brain barrier injury by C7-1080 dye is consistent with the traditional method, indicating the accuracy of C7-1080 in labeling the disruption of the blood-brain barrier.

[0121] As shown in Figure 47 a and b below, C7-1080 dye can evaluate the disruption of the blood-brain barrier after brain injury of different degrees. The evaluation results of C7-1080 dye on blood-brain barrier injury are consistent with the traditional method, indicating the accuracy of C7-1080 in evaluating the degree of disruption of the blood-brain barrier.

[0122] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent.

Claims

1. A cyanine dye with high efficiency in situ targeting of albumin in the near-infrared second region, characterized in that: The structural formula of the cyanine dye is: Where R structure is I or II: Here, n is any value from 2 to 10.

2. The cyanine dye according to claim 1, characterized in that When the R structure is I, the dye is Cx-808, x=n+2; when the R structure is II, the dye is Cx-1080, x=n+2.

3. The method for preparing a cyanine dye according to claim 1 or 2, characterized in that: The specific preparation steps are as follows: When the R structure is I and n=2, a mixture of compound a and ethyl 4-bromobutyrate is dissolved in acetonitrile and heated at reflux temperature for overnight reaction to obtain compound b; compound b is added to an aqueous solution of hydrogen bromide and heated at reflux temperature for 4 hours to generate compound c; compound c, compound d and anhydrous sodium acetate are dissolved in ethanol and stirred at 50° C. for overnight reaction to generate cyanine dye C4-808; wherein the structural formulas of compound a, compound b, compound c and compound d are as follows: When the R structure is I and n=3-10, compound a and compound e are mixed and heated at reflux temperature for overnight reaction to obtain compound c; compound c, compound d and anhydrous sodium acetate are dissolved in ethanol, stirred and reacted at 80° C. overnight to generate cyanine dye Cx-808, where x=5-12; wherein the structural formula of compound e is as follows: When the R structure is II, compound f and compound i react in N,N-dimethylformamide under the catalysis of potassium carbonate to generate compound g; compound g is mixed with HCl and reacted at 100°C to generate compound h; compound h, compound d and 2,6-di-tert-butyl-4-methylpyridine are dissolved in a mixed solution of toluene and n-butanol, and reacted at 100°C to generate cyanine dye Cx-1080; wherein the structural formulas of compound i, compound f, compound g and compound h are as follows:

4. Use of the cyanine dye prepared by the preparation method of the cyanine dye according to claim 3 in the preparation of biomimetic fluorescent protein.

5. A method for preparing a biomimetic fluorescent protein, characterized in that: The bionic fluorescent protein is a stable covalent complex formed by a cyanine dye prepared by the preparation method of the cyanine dye according to claim 3 and serum albumin, and the specific preparation method is as follows: Method 1: Dissolve human serum albumin in phosphate buffer solution to form an albumin solution, and add 10 μM cyanine dye Cx-1080; place the mixed system in a light-proof constant temperature shaker and incubate at room temperature to 60°C for 0-24 hours; after the incubation is completed, separate the mixture by 12% SDS-PAGE electrophoresis; Method 2: 200 μL of 600 μM cyanine dye Cx-1080 was injected into mice through the tail vein. The cyanine dye and serum albumin formed biomimetic fluorescent protein in situ in a physiological environment.

6. The method for preparing the biomimetic fluorescent protein according to claim 5, characterized in that: In the method 1 and the method 2, the value of x in the cyanine dye Cx-1080 is 7, that is, n=5; in the method 1, the optimal incubation temperature is 37-60° C., and the optimal incubation time is 0-12 h.

7. Use of the bionic fluorescent protein prepared according to the method for preparing the bionic fluorescent protein according to claim 5 or 6 in preparing a vascular imaging agent.

8. Use of the bionic fluorescent protein prepared according to the method for preparing the bionic fluorescent protein according to claim 5 or 6 in preparing a reagent for monitoring tissue barrier damage.

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