A near-infrared fluorescent probe and its preparation method and application

Abstract

The present invention discloses a near-infrared fluorescent probe and its preparation method and application. The chemical structural formula of the fluorescent probe is shown in Formula (3): It solves the problem that the influence of cell viscosity on the response characteristics of the tcf probe is ignored. When applied, at low viscosity, the intramolecular rotating bond of the probe can rotate at a high speed and the fluorescence is very weak. However, as the viscosity increases, the rotation process is blocked to promote the intramolecular charge transfer process. Therefore, tcf-vis1 emits a strong near-infrared fluorescence signal. Therefore, viscosity can be quantitatively monitored through an obvious "turn-on" fluorescence signal.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to a near-infrared fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] A stable intracellular microenvironment is an important condition for maintaining life activities such as cell proliferation, differentiation, and metabolism. Viscosity, as an important parameter of the intracellular microenvironment, plays a crucial role in the transport, interaction of biomolecules, and related life processes. Abnormal intracellular viscosity is closely related to various diseases, such as inflammation, Alzheimer's disease, cancer, etc. Therefore, the detection of intracellular viscosity is of great significance for the diagnosis and pathological research of diseases. However, due to the complexity and real-time nature of viscosity parameters, the detection of viscosity parameters in living systems poses great challenges. Traditional viscometer methods mainly include rotational viscometers, falling ball viscometers, and capillary viscometers. However, these methods are mainly based on in vitro viscosity rather than at the cell level and cannot provide the required spatial and temporal resolution. Therefore, there is an urgent need to develop a new method for detecting intracellular viscosity.

[0003] In recent years, fluorescent probes have become important tools for monitoring viscosity changes in biological systems due to their advantages such as simple operation, high sensitivity, real-time monitoring, and in-situ non-destructive imaging. For most viscosity probes, the mechanism of action is manifested as intramolecular charge transfer (TICT). These probes usually consist of a donor and an acceptor, forming a "D-π-A" molecular structure. Although many viscosity-sensitive fluorescent probes have been reported, these probes sometimes have some disadvantages, including low response signals, small Stokes shifts, and most probes are difficult to be used for in vivo animal imaging due to short emission wavelengths. Therefore, it is of great significance to develop responsive and specific near-infrared probes with large Stokes shifts for monitoring viscosity changes.

[0004] 2-Dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF), as an excellent electron-withdrawing fluorescent group, has attracted much attention due to its good photostability and super-resolution imaging. In recent years, various TCF-based fluorescent probes have been reported for bioimaging. However, in previous studies, except for a few exceptions, the influence of cell viscosity on the response characteristics of TCF probes has been ignored. Summary of the Invention

[0005] The present invention provides a near-infrared fluorescence probe, a preparation method and an application thereof, which solve the problem that the influence of cell viscosity on the response characteristics of the tcf probe is ignored in the prior art. When it is applied, at low viscosity, the intramolecular rotating bond of the probe can rotate at a high speed and the fluorescence is very weak. However, as the viscosity increases, the rotation process is blocked to promote the intramolecular charge transfer (ICT) process. Therefore, tcf-vis1 emits a strong near-infrared fluorescence signal. Therefore, viscosity can be quantitatively monitored through an obvious "turn-on" fluorescence signal.

[0006] To solve this technical problem, the present invention provides the following technical solutions:

[0007] A near-infrared fluorescence probe, the chemical structural formula of the fluorescence probe is shown in formula (3):

[0008]

[0009] A preparation method of the near-infrared fluorescence probe as described above, comprising the following steps:

[0010] S1. Prepare compound 1 shown in the following formula (1):

[0011]

[0012] S2. Prepare compound 2 shown in the following formula (2):

[0013]

[0014] S3. Prepare the compound shown in formula (3): Use a mixed solvent of tetrahydrofuran and ethanol as the reaction solvent, add compound 1, compound 2 and ammonium acetate; react at room temperature, after the reaction is completed, spin-dry the solvent, and separate to obtain the compound of formula (3).

[0015] Preferably, the preparation method of compound 1 shown in formula (1) is as follows:

[0016] Add 3.0 g of 4-bromobenzaldehyde and 2.7 g of 4-hydroxyphenylboronic acid solution to 60 mL of THF, add 15 mL of 2M K2CO3 and tetrakis(triphenylphosphine)palladium; stir the reaction mixture under a nitrogen atmosphere at 60 °C for 8 h, then pour the reaction mixture into 30 mL of water, extract the obtained mixture with 50 mL of DCM, wash with brine, dry with anhydrous Na2SO4, evaporate to dryness to obtain an oily substance, and purify by column chromatography to obtain the compound of formula (1).

[0017] Preferably, the preparation method of compound 2 shown in formula (2) is as follows:

[0018] Dissolve 200.0 mg of Na in 20 mL of absolute ethanol; stir at room temperature for 0.5 h; then, add 5.0 g of 3-hydroxy-3-methyl-2-butanone and 8.0 g of malononitrile, and stir at room temperature for 1.5 h; next, add 20 mL of absolute ethanol, and reflux for 1 h; after the reaction is completed, cool, filter, wash with cold ethanol, and dry under vacuum to obtain the compound of formula (2).

[0019] Preferably, the preparation method of the compound shown in formula (3) is as follows:

[0020] Add 2.2 g of compound 1 and 2.0 g of compound 2 to 20 mL of THF / EtOH, and add 780 mg of ammonium acetate; react at room temperature for 1.5 h, after the reaction is completed, spin-dry the solvent to obtain a solid, and purify by column chromatography on silica gel with a dichloromethane / methanol solvent eluent to obtain the compound shown in formula (3).

[0021] Preferably, the volume ratio of tetrahydrofuran to ethanol is 4:1.

[0022] This solution also provides the application of the above near-infrared fluorescent probe in detecting the viscosity of living cells.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This solution designs and prepares a red fluorescent probe TCF-vis1 based on the TCF skeleton for viscosity detection. At low viscosity, the intramolecular rotating bond of the probe can rotate at high speed and the fluorescence is very weak. However, as the viscosity increases, the rotation process is blocked, promoting the intramolecular charge transfer (ICT) process, so tcf-vis1 emits a strong near-infrared fluorescence signal. Therefore, the viscosity can be quantitatively monitored through an obvious "turn-on" fluorescence signal.

[0025] In addition, TCF-VIS1 has a large Stokes shift (184 nm) and a near-infrared emission of 644 nm, which can better eliminate background interference. At the same time, the probe has the advantages of high photostability, good sensitivity and selectivity.

[0026] Utilizing its excellent fluorescence properties, the TCF-VIS1 probe can successfully be used to detect the viscosity changes of living cells, and achieve viscosity fluorescence imaging of inflamed mice and tumor-bearing mice. This will contribute to promoting cancer diagnosis research and provide a potential tool for non-invasive diagnosis of viscosity-related diseases. Description of the Drawings

[0027] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not limit the embodiments of the present invention. In the drawings:

[0028] Figure 1 This is the synthetic route diagram of the TCF-VIS1 probe of the present invention;

[0029] Figure 2 This is for the 1 1H-NMR (400 MHz, DMSO-d6) spectrum of TCF-VIS1 of the present invention;

[0030] Figure 3 This is for the 13 13C-NMR (101 MHz, DMSO-d6) spectrum of TCF-VIS1 of the present invention;

[0031] Figure 4 (A) Absorption spectra of TCF-VIS1 (10 μM) in PBS and glycerol; Figure 4 (B) Fluorescence spectra of TCF-VIS1 (10 μM) in PBS and glycerol; Figure 4 (C) Fluorescence spectra of TCF-VIS1 (10 μM) at different glycerol fractions; Figure 4 (D) Linear relationship between log I644nm and log η in the viscosity range of 1.81 cp to 163.6 cp; Figure 4 (E) Fluorescence intensities of 10 μM TCF-VIS1 at 644 nm for different species (100 μM), (1) PBS; (2) NO 2- ; (3) K + ; (4) Mg 2+ ; (5) Ca 2+ ; (6) HCO3 - ; (7) Fe 3+ ; (8) Glu; (9) Cys; (10) BSA; (11) GSH; (12) urea; (13) H2S; (14) H2O2; (15) glycerol; Figure 4 (F) Photostability of the probe TCF-VIS1 in glycerol and PBS after continuous irradiation for 100 minutes (λex = 460 nm, λem = 644 nm);

[0032] Figure 5 (A) Absorption spectra of TCF-VIS1 (10 μM) of the present invention in different solvents; Figure 5 (B) Fluorescence spectra of TCF-VIS1 (10 μM) in different solvents (λex = 460 nm);

[0033] Figure 6 (A) Fluorescence spectra of PBS buffer solution of TCF-VIS1 (10 μM concentration) of the present invention at different pH values, λex = 460 nm; Figure 6(B) Fluorescence spectra of 60% glycerol of TCF-VIS1 (10 μM concentration) of the present invention at different pH values, λex = 460 nm; Figure 6 (C) Influence of pH on the fluorescence intensity of the TCF-VIS1 probe (10 μM concentration) at 644 nm in PBS buffer and 60% glycerol under the conditions of fG = 60% and λex = 460 nm; Figure 6 (D) Influence of temperature on the fluorescence intensity of the probe TCF-VIS1 (10 μM concentration) under the conditions of fG = 60% and λex = 460 nm;

[0034] Figure 7 (A) Optimized geometry of TCF-VIS1 of the present invention in the excited state; Figure 7 (B) Frontier molecular orbitals of TCF-VIS1 with dihedral angles of 0° and 90° calculated near C8-C13-C14-C15 respectively;

[0035] Figure 8 Cytotoxicity assay of tcf-vis1 of the present invention on Hela cells and HepG-2 cells incubated for 24 h;

[0036] Figure 9 Photostability of TCF-VIS1 (10 μM) in HeLa cells under continuous irradiation of the present invention, λex = 488 nm, λem = 600 - 750 nm, scale bar: 20 μm;

[0037] Figure 10 Study on fluorescence confocal imaging of TCF-VIS1 and Tracker on HepG-2 cells of the present invention; among them, (a, e) cells with confocal images (green channel) of Mit-Tracker green (50 nM) and Lyso-Tracker green (50 nM); (b, f) confocal images of cells with TCF-VIS1 (10 μM) (red channel); (c, g) overlay of the green channel and the red channel; (d, h) Pearson colocalization coefficients of TCF-VIS1 and Mito-Tracker green or Lyso-Tracker green; scale bar: 20 μm;

[0038] Figure 11The present invention uses TCF-VIS1 and Tracker for fluorescence confocal imaging of Hela cells; among them, (a, e) are cells with confocal images (green channels) of Mito-Tracker Green (50 nM) and Lyso-Tracker Green (50 nM) respectively; (b, f) are confocal images of cells with TCF-VIS1 (10 μM) (red channel); (c, g) are the overlays of the green channel and the red channel; (d, h) are the Pearson colocalization coefficients of TCF-VIS1 and Mito-Tracker Green or Lyso-Tracker Green; scale bar: 20 μm;

[0039] Figure 12 (A) is the confocal laser fluorescence image of HepG-2 cells of the present invention; among them, (a-c) incubate HepG-2 cells with TCF-VIS1 (10 μM) for 10 minutes; (d-f) incubate HepG-2 cells with nystatin (10 μM) for 45 minutes and then incubate with TCF-VIS1 (10 μM) for another 10 minutes; (g-i) incubate HepG-2 with dexamethasone (10 μM) for 45 minutes and then incubate with VIS-YW1 (10 μM) for another 10 minutes, Λex = 488 nm, λem = 600 - 750 nm, scale bar: 20 μm; Figure 12 (B) is the fluorescence intensity of the images (b), (e), (h) obtained by Image-J;

[0040] Figure 13 (A) is the confocal laser fluorescence image of Hela cells of the present invention; among them, (a-c) incubate Hela cells with TCF-VIS1 (10 μM) for 10 minutes; (d-f) incubate Hela cells with nystatin (10 μM) for 45 minutes and TCF-VIS1 (10 μM) for another 10 minutes, (g-i) incubate Hela with dexamethasone (10 μM) for 45 minutes and then incubate with VIS-YW1 (10 μM) for another 10 minutes, Λex = 488 nm, λem = 600 - 750 nm, scale bar: 20 μm; Figure 13 (B) is the fluorescence intensity of the images (b), (e), (h) obtained by Image-J;

[0041] Figure 14 (A) is the CLSM image of live HepG-2 cells treated with different concentrations of nystatin of the present invention, and then stained with TCF-VIS1 (10 μM) for 10 minutes; Figure 14 (B) is the fluorescence intensity of the images obtained from (A); Λex = 488 nm, λem = 600 - 750 nm, scale bar: 20 μm;

[0042] Figure 15(A) CLSM images of live HepG-2 cells treated with different concentrations of LPS in the present invention, and then stained with TCF-VIS1 (10 μM); Figure 15 (B) Fluorescence intensity of the images obtained from (A); Λex = 488 nm, λem = 600 - 750 nm; Scale bar: 20 μm;

[0043] Figure 16 (A) Imaging of normal cells (L929 and HL7702) and cancer cells (Hela, H596 and HepG-2) in the present invention with the probe TCF-VIS1 (10 μM) stained for 10 minutes, Scale bar: 20 μm, (λex = 488 nm; λem = 600 - 750 nm); Figure 16 (B) Using HepG-2 cells incubated with TCF-VIS1 (10 μM) for 10 minutes as a control, treating HepG-2 cells with erastin (10 μM) or erastin and Fer-1 (15 μM) for 6 hours, and then treating with TCF-VIS1 (10 μM) for another 10 minutes, Figure 16 (C) Relative fluorescence intensity of different cells in Figure A; Figure 16 For the relative fluorescence intensity of the images in Figure B Scale bar: 20 μm, (λex = 488 nm; λem = 600 - 750 nm),

[0044] Figure 17 CLSM images of normal and cancer cells stained with TCF-VIS1 (10 μM); Λex = 488 nm, λem = 600 - 750 nm. Scale bar: 50 μm, CLSM imaging was performed using a 20x objective;

[0045] Figure 18 (A) In vivo fluorescence imaging of TCF-VIS1 (50 μM, 100 μL) in (a) normal mice and (b) LPS-mediated inflammatory mice respectively, Figure 18 (B) Images of (a) normal and (b) tumor-bearing mice after injection of TCF-VIS1 (50 μM, 100 μL), Λex / λem = 470 / 700 nm,

[0046] Figure 19 Response mechanism of the TCF-VIS1 probe of the present invention to viscosity. Detailed implementation mode

[0047] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0048] Example 1

[0049] 1. Synthesis of Compound 1

[0050] A solution of 4 - bromobenzaldehyde (3.0 g, 16.2 mmol) and 4 - hydroxyphenylboronic acid (2.7 g, 19.5 mmol) was added to 60 mL of THF, along with 2 M K2CO3 (15 mL) and tetrakis(triphenylphosphine)palladium (940 mg, 5% mmol). The reaction mixture was stirred at 60 °C under a nitrogen atmosphere for 8 h. Then, the reaction mixture was poured into 30 mL of water, and the resulting mixture was extracted with DCM (50 mL × 3), washed with brine, dried over anhydrous Na2SO4, and evaporated to dryness to obtain an oil. The oil was purified by column chromatography (EA / PE = 1 / 20 - 1 / 10) to obtain Compound 1 (2.4 g) with a yield of 75%.

[0051] 2. Synthesis of Compound 2

[0052] Na (200.0 mg, 8.6 mmol) was dissolved in anhydrous ethanol (20 ml). The mixture was stirred at room temperature for 0.5 h. Then, 3 - hydroxy - 3 - methyl - 2 - butanone (5.0 g, 58.6 mmol) and malononitrile (8.0 g, 120 mmol) were added, and the mixture was stirred at room temperature for 1.5 h. Next, anhydrous ethanol (20 mL) was added, and the mixture was refluxed for 1 h. After the reaction was completed, it was cooled, filtered, washed with cold ethanol, and dried under vacuum to obtain Compound 2 (6.3 g) with a yield of 65%. 3. Synthesis of TCF - VIS1

[0053] A solution of Compound 1 (2.2 g, 11 mmol) and Compound 2 (2.0 g, 10 mmol) was added to THF / EtOH (4:1, 20 mL), and ammonium acetate (780 mg, 10 mmol) was added. The reaction was carried out at room temperature for 1.5 h. After the reaction was completed, the solvent was evaporated to dryness to obtain a solid, which was purified by column chromatography on silica gel using dichloromethane / methanol (v / v, 100:1) as the eluent to obtain Compound TCF - VIS1 (2.1 g) with a yield of 55%.

[0054] The synthesized TICT - based near - infrared viscosity - responsive fluorescent probe TCF - VIS1 uses TCF as the electron acceptor and 4 - hydroxybiphenyl as the electron donor. The strong push - pull effect and extended conjugated structure are beneficial to the emission of the probe in the near - infrared region and exhibit a large Stokes shift. At the same time, the freely rotating single bond is beneficial to fluorescence quenching at low viscosity, while at high viscosity, the rotation is restricted and the fluorescence emission is enhanced. The detailed synthesis process of TCF - VIS1 is as Figure 1 shown, as well as the 1H - NMR structure diagram ( Figure 2 ), 13C - NMR ( Figure 3 ). As can be seen from the figures:

[0055] The NMR data of Compound 1 are as follows: 1 1H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 4.95 (s, 1H).

[0056] The NMR data of Compound 2 are as follows: 1 1H NMR (400 MHz, CDCl3) δ 2.46 (s, 3H), 1.79 (s, 6H).

[0057] The NMR data of Compound 3 are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 16.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.24, (d, J = 16.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 2H), 1.81 (s, 6H).

[0058] 13 13C NMR (101 MHz, DMSO-d6) δ 177.60, 175.67, 158.65, 147.61, 144.20, 132.85, 130.70 (2C), 129.76, 128.60 (2C), 126.81 (2C), 116.41 (2C), 115.04, 113.23, 112.39, 111.47, 99.83, 99.21, 54.63, 25.62 (2C).

[0059] Example 2

[0060] Viscosity measurement and fluorescence spectroscopy analysis: First, a stock solution (1.0 mM) of TCF-VIS1 was prepared in DMSO. A 1 mL culture solution containing the stock solution (10 μL) and a solvent mixture (990 μL, water-glycerol solvent system) was prepared to obtain a final TCF-VIS1 concentration of 10.0 μM, and viscosity measurement was carried out. These solutions were measured with a fluorescence spectrometer, λex / em = 460 / 644 nm.

[0061] The relationship between the fluorescence emission intensity of the probe and the solvent viscosity can be represented by the following equation:

[0062] logi = C + xlogη

[0063] where η is the viscosity, I is the emission intensity, C is a constant, and x is the sensitivity of the probe to viscosity.

[0064] As Figure 4 shown in A, the absorption wavelength of TCF-VIS1 in aqueous solution is 405 nm, and in glycerol is 460 nm. The probe shows little fluorescence in PBS, while a significant fluorescence enhancement is obtained at 644 nm in glycerol ( Figure 4 B). The Stokes shift reaches 184 nm, which can effectively reduce the interference of the excitation light. In addition, the fluorescence intensity of TCF-VIS1 at 644 nm also increases as the glycerol component (fG) gradually increases from 0% to 99% ( Figure 4 C). As the viscosity increases from 0% (0.89 cP) to 99% (856 cP), the fluorescence intensity of TCF-VIS1 at 644 nm increases by 78-fold, indicating that TCF-VIS1 is highly sensitive to viscosity. In addition, in the viscosity range of 1.81 - 163.6 cP (R2 = 0.9919) ( Figure 4 D), the fluorescence intensities of TCF-VIS1 (logI644) and viscosity (logη) show a good linear relationship fitted by the equation (log I = 0.8949logη + 0.6305). The results show that the TCF-VIS1 probe can be used for quantitative measurement of viscosity. In addition, as Figure 4 shown in E, except for glycerol, TCF-VIS1 shows little or almost no response to other biologically relevant substances. These results indicate that TCF-VIS1 has high viscosity sensitivity and selectivity and is not interfered by other substances. In addition, the photostability of TCF-VIS1 was studied ( Figure 4 F), and the results show that the probe has good photostability in glycerol solution.

[0065] Then, we evaluated the UV-visible absorption and fluorescence spectral properties of TCF-VIS1 in different polar solvents (including PBS, ethanol, ethyl acetate, DMF, DMSO, methanol, acetonitrile, and glycerol). As shown in Figure 5 A, Figure 5 B and Table 1, Table 1 shows the spectral property data of the TCF-VIS1 probe in different solvents. As expected, the fluorescence of TCF-VIS1 shows relatively weak emission in all solvents except glycerol, and the fluorescence quantum yield is between 0 - 0.4%. In glycerol, the fluorescence emission is enhanced at 644 nm, and the fluorescence quantum yield is 0.97%. We also detected the fluorescence response of TCF-VIS1 by changing the temperature of glycerol (fG = 60%). When the temperature increases from 25 °C to 45 °C, the fluorescence intensity of TCF-VIS1 decreases regularly ( Figure 6D), indicating that the increase in temperature decreased the viscosity of the medium and reduced the fluorescence intensity. Next, the effect of pH on TCF-VIS1 was recorded at different pH values at the emission wavelength of 644 nm in PBS and 60% fG. As Figure 6 shown in A-C, in the range of pH 4.0 - 10.0, the fluorescence intensity of TCF-VIS1 did not change significantly, indicating that the fluorescence intensity of TCF-VIS1 was not affected by the pH value. These results suggest that TCF-VIS1 can be used as a viscosity-sensitive fluorescent probe in complex biological systems.

[0066] Table 1

[0067]

[0068] a Viscosity of the solvent. b Quantum yield was measured using steady-state and transient fluorescence spectrometers, in %. c Fluorescence was not observed

[0069] The structure of TCF-VIS1 was optimized using DFT / TDDFT at B3LYP / 6-31G in Gaussian 09. As Figure 7 shown, when the dihedral angle of C8-C13-C14-C15 was 0°, the probe TCF-VIS1 maintained a planar conformation with an oscillator strength of Fem 1.3868. However, when the dihedral angle of C8-C13-C14-C15 was 90°, the orthogonal conformation of the probe remained in the TICT state with an oscillator strength of only 0.0005 and weak fluorescence emission. These results indicate that the probe can form the TICT state through intramolecular rotation and produce weak non-fluorescent emission. These theoretical results clarify the electron cloud distribution of the TCF-VIS1 probe and help to deepen the understanding of its response mechanism. Obviously, TCF-VIS1 can be used as a probe for detecting viscosity changes.

[0070] Example 3

[0071] Cell imaging viscosity change: HepG-2 cells and Hela cells were grown in DMEM and 1640 medium supplemented with 10% fetal bovine serum (FBS) respectively in a 5% CO2 atmosphere at 37°C. The cells were seeded in glass-bottom culture dishes and cultured overnight. Then, the cells were co-cultured with dexamethasone or nystatin (10 μM) for 30 minutes, treated with the probe TCF-VIS1 (10 μM) at 37°C for 10 minutes, and then washed three times with PBS. Fluorescence imaging was performed using a confocal laser scanning microscope (CLSM). Λex = 488 nm, λem = 600 - 750 nm. Cancer cells (HeLa, H596, HepG-2) and normal cells (HL-7702 cells and L929) were incubated with TCF-VIS1 (10 μM) for 10 minutes. After washing with PBS, the cells were imaged on a CLSM (λex = 488 nm, λem = 600 - 750 nm).

[0072] Co-localization imaging experiment: The cells were incubated with TCF-VIS1 (10 μM) for 10 minutes and washed three times with PBS before use. The cell mitochondria were stained with a mitochondrial green fluorescent probe (50 nM), λex = 488 nm, λem = 515 nm. The cell lysosomes were stained with a lysosome green probe tracker (50 nM), λex = 488 nm, λem = 511 nm. Then, fluorescence imaging was captured on a CLSM.

[0073] The cytotoxicity of the probe in living HepG-2 cells and Hela cells was detected by the MTT method. The results showed that at a concentration of 40 μM, the cell survival rate was greater than 83%, indicating that the probe had low cytotoxicity( Figure 8 ). In addition, the photostability of TCF-VIS1 in cells was also studied. The results showed that the probe exhibited good photostability at the fluorescence imaging level( Figure 9 ).

[0074] The targeting ability of TCF-VIS1 to organelles was demonstrated by co-localization imaging of mitochondria and lysosomes. As Figure 10 shown in a - h, TCF-VIS1 overlapped with mitochondria and lysosomes in HepG-2 cells, resulting in similar co-localization imaging, and the Pearson co-localization coefficients (PC) were 0.79 and 0.81 respectively. Similar co-localization results were further confirmed in Hela cells( Figure 11 a - h), indicating that TCF-VIS1 could be retained in lysosomes and mitochondria.

[0075] TCF-VIS1 was used to detect changes in cell viscosity. Dexamethasone and nystatin are well-known ionophores that can enhance the viscosity of lysosomes and mitochondria. Therefore, dexamethasone and nystatin were selected as stimulants to detect the response of TCF-VIS1 to cell viscosity. As Figure 12 shown, HepG-2 cells were incubated with TCF-VIS1 alone for only 10 minutes, and the fluorescence was weak. When HepG-2 cells were pretreated with nystatin or dexamethasone and then incubated with TCF-VIS1, the fluorescence in the red channel was significantly enhanced ( Figure 12 A-B). Similarly, the TCF-VIS1 probe was also able to monitor the viscosity fluctuations of Hela cells after treatment with nystatin and dexamethasone ( Figure 13 A-B). These results indicate that TCF-VIS1 can be used to monitor changes in intracellular microviscosity. At the same time, HepG-2 cells were incubated with nystatin at different concentrations (0, 10, 40, 80 μM) for 30 minutes and then incubated with TCF-VIS1. As Figure 14 shown in A-B. As the concentration of nystatin increased, the fluorescence in the red channel gradually enhanced. This phenomenon indicates that the probe can be used to detect the viscosity fluctuations caused by nystatin. Since lipopolysaccharide is a typical inflammatory inducer, it can induce cell inflammation and lead to an increase in cell viscosity. Therefore, LPS was selected as a stimulant to further verify the imaging ability of TCF-VIS1 for changes in cell viscosity. In Figure 15 A-B, HepG-2 cells were pretreated with different concentrations of LPS for 30 minutes and then incubated with TCF-VIS1, and the fluorescence in the red channel increased significantly. These findings further indicate that the TCF-VIS1 probe is an effective tool for detecting changes in cell viscosity.

[0076] Previous studies have shown that cancer cells are more viscous than normal cells. Therefore, normal cells (L929, HL7702) and cancer cells (Hela, H596, HepG-2) were stained with TCF-VIS1 due to their ability to monitor viscosity. As Figure 16 shown in A and Figure 17 as shown, under the same imaging conditions, it was found that the fluorescence intensity of the probe was stronger than that of normal cells, indicating that TCF-VIS1 can distinguish cancer cells from normal cells. Therefore, this obvious difference indicates that TCF-VIS1 has the potential to diagnose cancer by detecting viscosity.

[0077] Using the high viscosity sensitivity of TCF-VIS1, the viscosity changes of living cells during iron concentration were studied. The study shows that during iron concentration, the cell viscosity increases due to the conversion of unsaturated lipids to lipid peroxides. The effect of the iron concentration activator Erastin on the viscosity of HepG-2 cells was studied. As Figure 16As shown in B-D, after treatment with only 10 μM TCF-VIS1 for 10 minutes, HepG-2 cells showed weak red fluorescence. After treatment with 10 μM erastin and 10 μM TCF-VIS1, the fluorescence in the red channel increased by approximately 3.8-fold. In addition, HepG-2 cells were treated with 10 μM erastin and 15 μM Fer-1 (iron concentration inhibitor) simultaneously, and then treated with 10 μM TCF-VIS1. Compared with untreated cells, the fluorescence of HepG-2 cells showed almost no change, indicating that the viscosity did not change significantly during the inhibition of iron concentration. Therefore, the probe can be used to detect the iron concentration process because the cell viscosity increases during the iron concentration process.

[0078] Example 4

[0079] In vivo fluorescence imaging: All animal procedures were carried out in accordance with the guidelines of the Experimental Animal Ethics Committee of Guangxi Normal University. Female BABL / c nude mice (8 weeks old) were purchased from Hunan SJA Experimental Animal Co., Ltd. The mice were intraperitoneally injected with LPS (1 mg, 500 μL normal saline). After 6 hours, the mice were intraperitoneally injected with TCF-VIS1 (50 μM, 100 μL). As a control, mice without LPS were only injected with TCF-VIS1 (50 μM, 100 μL). A suspension of 1×106 4T1 cells was subcutaneously implanted into the left posterior side of the mice. Seven days after inoculation, the mice were anesthetized during the experiment. TCF-VIS1 (50 μM, 100 μL) was intratumorally injected into the mice carrying 4T1 tumors. Fluorescence imaging was performed on an FXPRO in vivo imaging system (Bruker, Germany, λex = 470 nm, λem = 700 nm).

[0080] Inflammation increases the viscosity level in the body to a certain extent. Lipopolysaccharide, as a cell wall component of Gram-negative bacteria, is an effective reagent for establishing an inflammatory injury model. Therefore, a mouse inflammation model was established by intraperitoneal injection of LPS. In Figure 18 A, normal mice intraperitoneally injected with TCF-VIS1 showed insignificant fluorescence. At the same time, enhanced fluorescence signals were observed in the skin tissues of inflammatory mice. Therefore, we expect that the TCF-VIS1 probe can be used for the diagnosis of viscosity-related diseases.

[0081] It has been reported that the viscosity of tumor cells is higher than that of normal cells. Therefore, we subcutaneously injected 4T1 cells into the left posterior side of the mice to prepare a tumor-bearing mouse model and tested the response of TCF-VIS1 to viscosity changes in vivo. As Figure 18 shown in B, the fluorescence intensity of normal mice was weak when injected with TCF-VIS1, while the fluorescence intensity of tumor-bearing mice was strong when TCF-VIS1 was intratumorally injected, indicating that the TCF-VIS1 probe can be used as an effective tool for tumor visualization.

[0082] Figure 19 It shows the mechanism of the fluorescence probe TCF-vis1 for viscosity detection. At low viscosity, the intramolecular rotating bond of the probe can rotate at a high speed and the fluorescence is very weak. However, as the viscosity increases, the rotation process is blocked to promote the intramolecular charge transfer (ICT) process, so tcf-vis1 emits a strong near-infrared fluorescence signal.

[0083] In summary, by utilizing the viscosity detection characteristics of TICT, a novel near-infrared fluorescence probe TCF-VIS1 with a large Stokes shift (184 nm) was prepared through a simple reaction. This probe has good sensitivity, selectivity, photostability and low cytotoxicity. In the water-glycerol system, the fluorescence of the probe at 644 nm increases significantly with the increase of viscosity. TCF-VIS1 has been successfully used for viscosity monitoring of tumor cells, normal cells, inflammatory mice and tumor mice with its excellent fluorescence characteristics. These results indicate that TCF-VIS1 can be used as an effective tool for detecting viscosity-related diseases. In addition, the influence of cellular microviscosity on the response characteristics of the TCF fluorescence probe should not be ignored in future research.

[0084] The specific embodiments described above further elaborate on the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only for the specific embodiments of the present invention and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Near-infrared fluorescence probe, characterized in that, The chemical structural formula of the fluorescent probe is shown in Formula (3):

2. A preparation method of the near-infrared fluorescence probe as described in claim 1, characterized in that, It includes the following steps: S1. Prepare compound 1 shown in the following Formula (1): S2. Prepare compound 2 shown in the following Formula (2): S3. Prepare the compound shown in Formula (3): Use a mixed solvent of tetrahydrofuran and ethanol as the reaction solvent, add compound 1, compound 2 and ammonium acetate; react at room temperature, after the reaction is completed, spin-dry the solvent, and separate to obtain the compound of Formula (3).

3. The preparation method of the near-infrared fluorescent probe according to claim 2, wherein The preparation method of compound 1 shown in Formula (1) is as follows: Add 3.0 g of 4-bromobenzaldehyde and 2.7 g of 4-hydroxyphenylboronic acid solution to 60 mL of THF, add 15 mL of 2M K2CO3 and tetrakis(triphenylphosphine)palladium; stir the reaction mixture under a nitrogen atmosphere at 60 °C for 8 h, then pour the reaction mixture into 30 mL of water, extract the obtained mixture with 50 mL of DCM, wash with brine, dry with anhydrous Na2SO4, evaporate to dryness to obtain an oily substance, and purify by column chromatography to obtain the compound of Formula (1).

4. The preparation method of the near-infrared fluorescence probe according to claim 2, wherein The preparation method of compound 2 shown in Formula (2) is as follows: Dissolve 200.0 mg of Na in 20 ml of absolute ethanol; stir at room temperature for 0.5 h; then, add 5.0 g of 3-hydroxy-3-methyl-2-butanone and 8.0 g of malononitrile, stir at room temperature for 1.5 h; next, add 20 mL of absolute ethanol and reflux for 1 h; after the reaction is completed, cool, filter, wash with cold ethanol, and dry in vacuo to obtain the compound of Formula (2).

5. The preparation method of the near-infrared fluorescent probe according to claim 2, wherein The preparation method of the compound shown in Formula (3) is as follows: Add 2.2 g of compound 1 and 2.0 g of compound 2 to 20 mL of THF / EtOH, add 780 mg of ammonium acetate; react at room temperature for 1.5 h, after the reaction is completed, spin-dry the solvent to obtain a solid, and purify by column chromatography on silica gel with a dichloromethane / methanol solvent eluent to obtain the compound shown in Formula (3).

6. The preparation method of the near-infrared fluorescent probe according to claim 2, wherein The volume ratio of tetrahydrofuran to ethanol is 4:1.

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