Polarity-sensitive lipid droplet fluorescence imaging probe molecule based on benzobithiophene tetraoxide receptor and application of polarity-sensitive lipid droplet fluorescence imaging probe molecule

Abstract

The invention discloses a polarity-sensitive lipid droplet fluorescence imaging probe molecule based on a benzobithiophene tetraoxide receptor and application of the polarity-sensitive lipid droplet fluorescence imaging probe molecule, and belongs to the technical field of biological imaging. The chemical name of the probe is 4-(6-(7-(diethylamino)-9, 9-dimethyl-9H-furan-2-yl)-4, 8-bis (2-methoxyethoxy)-1, 1, 5, 5-dibenzo [1, 2-b: 4, 5-b '] dithiabenzene-2-yl) benzenitrile (Lipi-PS), and the structural formula of the probe is shown in the specification. The invention also discloses an application of the fluorescent probe as a cell fluorescence imaging reagent, especially an application of the fluorescent probe in cell lipid droplet confocal, delayed three-dimensional confocal and hyperspectral fluorescence imaging. The fluorescent probe Lipi-PS disclosed by the invention is a lipid droplet fluorescent probe with the advantages of polarity hypersensitive emission characteristic, high lipid droplet labeling selectivity, high light stability, excellent biocompatibility and the like, and has a huge application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging technology, and particularly relates to a polarity-sensitive cell lipid droplet fluorescence imaging probe molecule based on a benzobithiophene tetraoxide receptor and its application as a cell fluorescence imaging reagent. Background Art

[0002] Biofluorescence imaging technology has the advantages of being non-invasive, highly sensitive, and having high spatiotemporal resolution, and is a core tool for biomedical research. Traditional fluorescence imaging techniques (such as widefield, confocal, two-photon, etc.) can observe the morphological structure of tissues, cells, and subcellular structures. Fluorescence lifetime imaging (FLIM), which has been developed in recent years, further gives the fluorescence lifetime of each pixel on the basis of traditional fluorescence imaging. By combining fluorescent probes whose fluorescence lifetime is sensitive to the microenvironment (polarity, viscosity, pressure, etc.), FLIM imaging can reflect the microenvironmental information of biological samples through changes in the length of fluorescence lifetime while observing the morphological structure. This adds a new dimension of information to traditional fluorescence imaging and is of great significance for biomedical research.

[0003] Similar to FLIM imaging, hyperspectral fluorescence imaging (HSFI) further enhances traditional fluorescence imaging by providing the fluorescence wavelength of each pixel. By using fluorescent probes whose fluorescence wavelengths are sensitive to the microenvironment, HSFI can also observe morphological structures and provide information about microenvironmental changes. HSFI imaging offers potential advantages over FLIM imaging in terms of imaging equipment requirements and imaging speed. However, the development of HSFI lags significantly behind FLIM, and its application in biological imaging has been rarely reported. This is primarily due to the fact that probes sensitive to the microenvironment by fluorescence lifetime are more mature, while probes sensitive to the microenvironment by fluorescence wavelength are relatively scarce and have extremely low sensitivity. For example, the classic microenvironmental polarity-sensitive probe Nile Red exhibits a fluorescence wavelength shift of only 7 nm when changing solvents from toluene to dioxane. However, the microenvironmental polarity shifts in biological systems are much smaller, resulting in even smaller fluorescence wavelength shifts, making HSFI detection of wavelength shifts extremely difficult. Therefore, the development of new fluorescent probes that are highly sensitive to microenvironmental changes is crucial for the application and development of HSFI imaging technology. Summary of the Invention

[0004] In view of the shortcomings of existing lipid droplet imaging fluorescent probe molecules in terms of polarity sensitivity, the problem to be solved by the present invention is to provide a polarity-sensitive lipid droplet fluorescent imaging probe molecule based on a benzobithiophene tetroxide receptor and its application as a cell fluorescence imaging reagent, especially in fluorescence imaging modes such as cell lipid droplet HSFI, where the fluorescence imaging is confocal fluorescence imaging, time-lapse three-dimensional confocal fluorescence imaging or hyperspectral fluorescence imaging (HSFI).

[0005] The present invention discloses a polarity-sensitive lipid droplet fluorescence imaging probe molecule based on a benzobithiophene tetraoxide receptor. Its chemical name is 4-(6-(7-(diethylamino)-9,9-dimethyl-9H-furan-2-yl)-4,8-bis(2-methoxyethoxy)-1,1,5,5-tetraoxydibenzo[1,2-b:4,5-b']dithiabenzene-2-yl)benzonitrile, abbreviated as Lipi-PS. This fluorescence imaging probe molecule has a donor-π bridge-acceptor structure, and its chemical structure is shown in Formula (I):

[0006]

[0007] The present invention prepares the fluorescent imaging probe molecule for the first time and applies it to fluorescent imaging modes such as HSFI. The preparation reaction formula of the fluorescent imaging probe molecule is as follows:

[0008]

[0009] In response to the shortcomings of existing cell lipid droplet fluorescence imaging probe molecules, the present invention selects a donor-π bridge-acceptor type fluorescent molecule based on the benzobithiophene tetraoxide receptor in the development of a new lipid droplet imaging fluorescent imaging probe molecule. In the present invention, a fluorene structure π bridge, a diethylamine donor, and a benzocyanide receptor are introduced on the basis of the benzobithiophene tetraoxide receptor structure. The prepared fluorescent molecule exhibits an ultra-large Stokes shift and a strong solvation effect, and has polar supersensitive emission characteristics. The dual acceptor action of benzocyanide and benzobithiophene tetraoxide significantly reduces the LUMO energy level, thereby improving the photostability of the fluorescent imaging probe molecule. The introduction of diethylamine provides a strong donor, and the introduction of fluorene provides a π bridge with a large conjugated area and free rotation. The introduction of an alkoxy chain on the central structure can adjust the hydrophilicity of the probe molecule, giving it a higher lipid droplet labeling selectivity. The polar supersensitive emission characteristics enable the probe to exhibit a large fluorescence wavelength change under different polarity environments. In fluorescence imaging applications, the ultra-high polarity sensitivity, high labeling selectivity, high photostability, and good biocompatibility enable this probe molecule to achieve high-fidelity confocal imaging of cellular lipid droplets, time-lapse three-dimensional confocal imaging, and HSFI. Therefore, the development of the fluorescent imaging probe molecule Lipi-PS provides a powerful tool for fluorescence imaging of cellular lipid droplets.

[0010] The cells described in the present invention are HeLa cells.

[0011] The cell lipid droplet fluorescence imaging probe molecule Lipi-PS prepared by the present invention is a multifunctional fluorescence imaging probe molecule that can be used for time-lapse three-dimensional confocal imaging and HSFI with polar ultrasensitive emission characteristics, high labeling selectivity, high photostability and excellent biocompatibility (Examples 6-9).

[0012] Experimental results confirm that the fluorescent imaging probe molecule Lipi-PS described in the present invention has ultra-polarity-sensitive emission characteristics. Its polarity-sensitive characteristics were confirmed by polarity-dependent fluorescence spectroscopy tests, and its excited-state polarity-dependent characteristics were verified by transient absorption tests. By comparison with the commercial lipid droplet dyes BODIPY and Nile Red, it was found that the probe molecule Lipi-PS can efficiently and selectively label cellular lipid droplets with a high imaging signal-to-noise ratio. Cytotoxicity tests confirmed that the probe molecule has good biocompatibility. In addition, the probe molecule also has high photostability and can be used for time-lapse three-dimensional confocal imaging and HSFI of lipid droplets. Therefore, Lipi-PS can be used as a lipid droplet fluorescent imaging probe molecule to study the complex physiological functions of cellular lipid droplets and promote the development of lipid droplet cell biology. The design strategy of this molecule has important guiding significance for the development of polarity-sensitive fluorescent imaging probe molecules.

[0013] In summary, the Lipi-PS described in this invention is a novel lipid droplet fluorescence imaging probe. Compared to other lipid droplet fluorescence imaging probes, its ultra-polarity-sensitive emission properties enable it to detect changes in lipid droplet polarity. Given these characteristics, its application in cellular lipid droplet fluorescence imaging has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : H NMR spectrum of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 of the present invention;

[0015] Figure 2 : Fluorescence spectra of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 of the present invention in solutions of different polarities (corresponding to Example 2);

[0016] Among them, the dotted line is the fluorescence spectrum in toluene solution, and the solid line is the fluorescence spectrum in dioxane solution;

[0017] Figure 3 : Femtosecond transient absorption spectrum of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 of the present invention in a dioxane solution (corresponding to Example 3);

[0018] Figure 4 : A bar graph showing the cell viability of HeLa cells stained with different concentrations of the fluorescent imaging probe molecule Lipi-PS for 24 hours (corresponding to Example 5);

[0019] Figure 5 : Photograph of colocalization of the fluorescent imaging probe molecule Lipi-PS and the commercial lipid droplet dye BODIPY in HeLa cells (corresponding to Example 6);

[0020] Figure 5(a) is a photograph of BODIPY in the 500-550nm imaging channel under 488nm laser excitation; Figure 5 Middle (b) is a photograph of Lipi-PS in the 650-750nm imaging channel under 560nm laser excitation; Figure 5 Middle (c) is the superposition of the first two fluorescence images and the bright field image; Figure 5 Middle (d) is the Pearson correlation coefficient (R=0.9) of the first two fluorescence channels (scale bar: 10 μm).

[0021] Figure 6 : Quantification of the signal-to-noise ratio of the fluorescent imaging probe molecule Lipi-PS and the commercial lipid droplet dyes BODIPY and Nile Red in HeLa cells (corresponding to Example 7);

[0022] Figure 6 (a) to (c) are confocal images of HeLa cells stained with different fluorescent imaging probe molecules, scale bar: 10 μm; Figure 6 (d) to (f) are Figure 6 Magnified images of the boxed areas in (a) to (c) and the signal-to-noise ratio of the selected areas (scale bar: 5 μm).

[0023] Figure 7 : Time-lapse three-dimensional confocal photograph of HeLa cells stained with the fluorescent imaging probe molecule Lipi-PS (corresponding to Example 8);

[0024] Among them, the left picture is a three-dimensional imaging photo at 0 minutes, and the right picture is a three-dimensional imaging photo at 240 minutes.

[0025] Figure 8 : HSFI photograph of HeLa cells stained with the fluorescent imaging probe molecule Lipi-PS (corresponding to Example 9);

[0026] Figure 8 Middle (a) is the HSFI photograph of the control group without stimulation (scale bar: 10 μm); Figure 8 Middle (b) is the HSFI photograph of the cholesterol-stimulated group (scale bar: 10 μm); Figure 8 Middle (c) is the HSFI photograph of the group stimulated with oleic acid (scale bar: 10 μm). DETAILED DESCRIPTION

[0027] Example 1:

[0028] Synthesis of 4-(6-(7-(diethylamino)-9,9-dimethyl-9H-furan-2-yl)-4,8-bis(2-methoxyethoxy)-1,1,5,5-tetraoxydibenzo[1,2-b:4,5-b']dithiabenzene-2-yl)benzonitrile (Lipi-PS)

[0029] The compound 2,6-dibromo-4,8-bis(2-methoxyethoxy)benzo[1,2-b:4,5-b']dithiophene 1,1,5,5-tetraoxide (0.650 g, 1.16 mmol), the compound N,N-diethyl-9,9-dimethyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9H-furan-2-amine (0.499 g, 1.28 mmol) and potassium carbonate (0.480 g, 3.48 mmol) were added to a mixed solution of toluene (32 mL), ethanol (4 mL) and water (4 mL), followed by the addition of tetrakistriphenylphosphine palladium (00.32 g, 0.028 mmol), and the resulting mixture was stirred at 90 °C for 12 hours. After cooling to room temperature, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (0.069 g, 0.30 mmol), potassium carbonate (0.084 g, 0.60 mmol) and tetrakistriphenylphosphine palladium (0.016 g, 0.014 mmol) were added, and the mixture was stirred at 90 ° C for 18 hours. After cooling to room temperature again, water (100 mL) was added and the mixture was extracted with dichloromethane (200 mL). The organic layer was washed with brine (50 mL), then dried over anhydrous magnesium sulfate and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain a black powder compound Lipi-PS (0.083 g, 0.11 mmol).

[0030] 1 H NMR (500MHz, CD2Cl2): δ7.99(s,1H),7.98–7.95(m,2H),7.86–7.82(m,3H),7.82–7.76(m,2H),7.69(d,J=7.3Hz,1H),7.64(d,J =7.6Hz,1H),6.80–6.69(m,2H),4.68–4.60(m,4H),3.89–3.84(m,4H),3.57–3.45(m,10H),1.55(s,6H),1.26(t,J=7.1Hz,6H).

[0031] Figure 1 This is the H-NMR spectrum of the fluorescent imaging probe molecule Lipi-PS synthesized in Example 1, indicating that the target product Lipi-PS was prepared.

[0032] Example 2: Determination of the fluorescence spectra of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 in different solutions

[0033] The fluorescent imaging probe molecule Lipi-PS synthesized in Example 1 was prepared into a 10 μM solution using toluene and dioxane solvents. The fluorescence spectrum was collected using an Ocean Optics fiber optic fluorescence spectrometer under the excitation of 470 nm excitation light, as shown in FIG. Figure 2 As shown, the dotted line is the fluorescence spectrum in toluene, and the solid line is the fluorescence spectrum in dioxane, indicating that the fluorescence spectrum of the fluorescent imaging probe molecule Lipi-PS is extremely sensitive to the polarity of the solvent, and a very small change in polarity will cause a significant red shift in the fluorescence spectrum.

[0034] Example 3: Determination of femtosecond transient absorption spectrum of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 in dioxane solution

[0035] The fluorescent imaging probe molecule Lipi-PS synthesized in Example 1 was prepared into a 10 μM solution using dioxane solvent. The femtosecond transient absorption spectrum in the range of 500 nm to 750 nm was measured by excitation with a 405 nm pump laser. The transient absorption data was processed using Origin software to obtain the transient absorption spectrum of Lipi-PS in dioxane. Figure 3 As shown in the figure, two signal bands appear, corresponding to ground-state photobleaching and excited-state absorption. Due to the polarity-dependent nature of the excited state, the stimulated emission signal is weakened, and the stimulated emission band that should appear is covered by the excited-state absorption, showing a significant difference from the non-polar solvent cyclohexane.

[0036] Example 4: Cell culture

[0037] All percentages in this embodiment are volume fractions.

[0038] HeLa cells were cultured in a 37°C, 5% CO2 incubator in high-glucose DMEM containing 10% fetal bovine serum and 1% double-streptomycin (penicillin-streptomycin mixture). The fetal bovine serum, double-streptomycin, and high-glucose DMEM were purchased directly from a biological reagent company.

[0039] After the cells grow to the logarithmic phase, we perform cell passage treatment: after aspirating the original 5mL culture medium in the cell culture flask, we use 2mL of high-glucose DMEM culture medium (containing 1% double antibody) without fetal bovine serum to wash the cell surface. After aspirating the culture medium, we use 0.5mL of trypsin to digest the cells for 2 minutes. After most of the cells are detached from the wall, we add 2mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody and blow evenly. Take an appropriate amount of the cell dispersion and transfer it to a new cell culture flask and culture dish respectively, and culture it in a CO2 cell culture incubator. After the cells in the culture dish reach an appropriate concentration, they are used for confocal or super-resolution imaging experiments.

[0040] Example 5: Test of cytotoxicity of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1

[0041] We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to test the cytotoxicity of the fluorescent imaging probe molecule Lipi-PS (HeLa cells). HeLa cells were seeded in 96-well plates (1*10 4 cells), and cultured them in a CO2 cell culture incubator for 24 hours. Afterwards, the culture medium of the middle 60 wells was replaced with culture medium containing different concentrations (0, 0.5, 1.0, 2.0, 5.0 and 10.0 μM) of fluorescent imaging probe molecules Lipi-PS and 1% (volume fraction) DMSO (10 parallel tests were set for each concentration). After another 24 hours of culture, MTT reagent (10 μL per well) was added to these wells, and the wells were returned to the cell culture incubator and continued to be cultured for 4 hours. After removing the original culture medium in these wells, DMSO (100 μL per well) was added to dissolve the generated formazan crystals. After standing at room temperature for 30 minutes, the absorbance of each well was measured at 530 nm using a microplate reader. Because only living cells can react with the MTT reagent to form formazan crystals, we can compare the average absorbance value of each group of wells with the average absorbance value of the control group (10 wells with a probe molecule concentration of 0) to calculate the cell survival rate. The results are as follows. Figure 4 As shown, it shows that the fluorescent imaging probe molecule Lipi-PS has basically no cytotoxicity, and the probe molecule at a concentration of 10.0 μM will not affect the normal growth of HeLa cells within 24 hours.

[0042] Example 6: Co-staining experiment of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 and the commercial lipid droplet dye BODIPY in HeLa cells

[0043] We cultured HeLa cells in a 20mm diameter glass-bottomed culture dish and propagated them in a CO2 incubator for 2 days. After taking them out of the incubator, the original DMEM culture medium in the culture dish was removed, and 1mL of DMEM culture medium containing Lipi-PS (2μM), BODIPY (2μM) and 1% (volume fraction) DMSO was added. The cells were then placed in a cell culture incubator and cultured for another 2 hours. After removal, they were washed three times with HBSS solution, and then fluorescence imaging was performed in HBSS solution. Figure 5 As shown, we can clearly observe that the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 and the commercial lipid droplet dye BODIPY can achieve good co-localization in HeLa cells, indicating that the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 has excellent cell lipid droplet labeling ability.

[0044] Example 7: Comparison of the imaging signal-to-noise ratio of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1 and the commercial lipid droplet dyes Nile Red and BODIPY in HeLa cells

[0045] After removing the three culture dishes filled with HeLa cells from the cell culture incubator in Example 4, the original DMEM culture medium was removed and DMEM culture medium (containing 1% DMSO) containing 2 μM Lipi-PS, 2 μM Nile Red, and 2 μM BODIPY was added. After returning to the incubator and continuing to culture for 2 hours, the three culture dishes were removed and washed three times with HBSS solution before fluorescence imaging. Figure 6 As shown, we selected areas in three culture dishes and zoomed in. By comparing the signal-to-noise ratio of the selected area within the zoomed area, we found that the fluorescent imaging probe molecule Lipi-PS exhibited an imaging signal-to-noise ratio much higher than that of Nile Red and BODIPY, indicating its ultra-high lipid droplet labeling selectivity.

[0046] Example 8: Time-lapse three-dimensional confocal imaging of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1

[0047] After removing the culture dish filled with HeLa cells from the incubator in Example 4, the original DMEM culture medium was removed and replaced with DMEM containing 2 μM Lipi-PS and 1% DMSO. The dish was returned to the incubator and incubated for 2 hours. Free probe molecules were washed away with HBSS solution, and oleic acid was added for stimulation. In situ time-lapse 3D confocal imaging was performed. Multiple scans were recorded at different Z-axis depths every half hour within the selected area. Three-dimensional reconstruction was then performed using LAS X software to obtain the following image. Figure 7 The three-dimensional confocal images shown are: the left image is the first group (0 minutes) of three-dimensional images, and the right image is the three-dimensional image after 240 minutes of oleic acid stimulation. The clear spatial distribution of lipid droplets fully verifies the high lipid droplet labeling selectivity and high photostability of the fluorescent imaging probe molecule Lipi-PS.

[0048] Example 9: HSFI of the fluorescent imaging probe molecule Lipi-PS prepared in Example 1

[0049] After we took the culture dish covered with HeLa cells in Example 4 out of the incubator, we removed the original DMEM culture medium, added DMEM culture medium containing 2μM Lipi-PS and 1% DMSO, put it back into the incubator and cultured for 2 hours, then took it out, washed it 3 times with HBSS solution and imaged it in HBSS solution. A total of three experimental groups were set up, namely the control group without any stimulation, the experimental group with cholesterol stimulation and the experimental group with oleic acid stimulation. After each experimental group used 560nm excitation light to excite the fluorescent imaging probe molecule in the selected area, it took fluorescence photos in 42 consecutive channels within the range of 570-780nm with 5nm as one channel. The data was then processed by Matlab software, and the spectral information of each pixel was superimposed on the intensity photo in the form of pseudo color, thereby realizing HSFI. From Figure 8 As can be seen in the figure, the fluorescence wavelength of lipid droplets in the control group is around 660nm, the fluorescence wavelength of the cholesterol-stimulated group is around 620nm, and the fluorescence wavelength of the oleic acid-stimulated group is around 680nm. This shows that applying different stimuli can change the internal polarity of cellular lipid droplets, and the polarity-sensitive properties of the fluorescent imaging probe molecule Lipi-PS can significantly amplify these tiny changes, with a maximum fluorescence wavelength change of 60nm. This fully verifies the polarity-sensitive properties of the fluorescent imaging probe molecule Lipi-PS.

Claims

1. A polarity-sensitive lipid droplet fluorescence imaging probe molecule based on benzobithiophene tetraoxide receptor, the structural formula of which is shown below:

2. Use of the polarity-sensitive lipid droplet fluorescence imaging probe based on benzobithiophene tetraoxide receptor according to claim 1 as a cell fluorescence imaging reagent.

3. Use of a polarity-sensitive lipid droplet fluorescence imaging probe based on a benzobithiophene tetraoxide receptor as claimed in claim 2 as a cell fluorescence imaging reagent, characterized in that: The cells are HeLa cells.

4. Use of a polarity-sensitive lipid droplet fluorescence imaging probe based on a benzobithiophene tetraoxide receptor as claimed in claim 2 or 3 as a cell fluorescence imaging reagent, characterized in that: Fluorescence imaging includes confocal fluorescence imaging, time-lapse three-dimensional confocal fluorescence imaging, or hyperspectral fluorescence imaging.

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