Yellow light emission fluorescent probe with polarity sensitive characteristic and application thereof in preparation of cell lipid droplet imaging reagent

Abstract

The invention discloses a yellow-light-emitting fluorescent probe with a polarity sensitive characteristic and application thereof in preparation of a cell lipid droplet imaging reagent, and belongs to the technical field of biological imaging. According to the present invention, the fluorescent probe Lipi-Yellow has excellent dyeing selectivity, and under the same dyeing and imaging conditions, the Lipi-Yellow shows the dyeing selectivity more excellent than the dyeing selectivity of the commercial lipid droplet fluorescent probe BODIPY and the dyeing selectivity of the commercial lipid droplet fluorescent probe Nile Red; therefore, the fluorescent probe Lipi-Yellow can be used for specifically marking the lipid droplets. The fluorescence imaging results provide abundant information about the lipid droplets, and the polarity change of the lipid droplets can be deeply understood by virtue of the polarity sensitive characteristic, so that the fluorescent probe Lipi-Yellow is a polarity sensitive lipid droplet fluorescent probe with high light stability and dyeing selectivity; the method can be used for marking lipid droplets in cells and monitoring the polarity change of the lipid droplets before and after oleic acid stimulation, and has a huge application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging technology, and specifically relates to a yellow light emitting fluorescent probe with polarity-sensitive characteristics and its application in preparing cell lipid droplet imaging reagents, which can be used to mark lipid droplets in cells and monitor changes in lipid droplet polarity before and after oleic acid stimulation. Background Art

[0002] Lipid droplets are the main organelles for storing lipids in eukaryotic cells. They are mainly composed of neutral lipids (cholesterol esters and triglycerides). As a highly dynamic organelle, lipid droplets can not only affect important cellular activities such as lipid metabolism, storage, and signal transduction, but are also closely related to protein degradation, energy homeostasis, cell differentiation, proliferation, and apoptosis. The study of lipid droplets is of great significance, and more and more studies have confirmed that the polarity of lipid droplets is one of the important microenvironmental parameters, and its dynamic changes can reflect the metabolic state of cells to a certain extent. Therefore, the study of lipid droplets is very necessary.

[0003] In order to observe and study lipid droplets, fluorescence microscopy such as laser scanning confocal microscopy is a powerful tool. The application of fluorescence imaging technology requires the use of fluorescent probes with excellent performance. Therefore, through reasonable molecular design, we synthesized the polarity-sensitive lipid droplet fluorescent probe Lipi-Yellow that emits yellow light. It is used as a cell lipid droplet imaging reagent to observe cell lipid droplets and monitor the changes in their polarity before and after oleic acid stimulation, providing a powerful tool for more in-depth research on lipid droplets. Summary of the invention

[0004] In view of the shortcomings of existing cell lipid droplet fluorescent probes, the problem to be solved by the present invention is to design and synthesize a yellow light emitting fluorescent probe with high polarity sensitivity characteristics having a large Stokes shift and good fluorescence quantum yield, so as to realize the visualization of cell lipid droplets and monitor the polarity change differences of the lipid droplet environment.

[0005] The yellow light emitting fluorescent probe Lipi-Yellow with polarity-sensitive characteristics described in the present invention is a donor-acceptor structure, and its structural formula is as follows:

[0006]

[0007] Its preparation reaction formula is as follows:

[0008]

[0009] The yellow light emitting fluorescent probe Lipi-Yellow with polarity-sensitive characteristics described in the present invention can be used in the preparation of cell lipid droplet imaging reagents, and can be used to mark lipid droplets in cells and monitor changes in lipid droplet polarity before and after oleic acid stimulation.

[0010] In view of the lack of selectivity of existing cell lipid droplet fluorescent probes for lipid droplet staining, the present invention adjusts the appropriate lipid-water partition coefficient when developing a new lipid droplet fluorescent probe, so that the fluorescent probe has good targeting ability for lipid droplets. In addition, by introducing a donor-acceptor structure constructed by triphenylamine and cyano, it has intramolecular charge transfer state fluorescence emission in a highly polar solvent environment. The highly polarity sensitive characteristics of the fluorescent probe emission can be used to monitor the polarity changes of lipid droplets.

[0011] The cells described in the present invention are HeLa cells.

[0012] The experimental results confirm that the fluorescent probe Lipi-Yellow of the present invention has excellent staining selectivity. Under the same staining and imaging conditions, Lipi-Yellow exhibits better staining selectivity than commercial lipid droplet fluorescent probes BODIPY and Nile Red. Therefore, the fluorescent probe Lipi-Yellow can specifically label lipid droplets. These fluorescence imaging results provide rich information about lipid droplets, and with the help of polarity-sensitive properties, we can gain an in-depth understanding of the polarity changes of lipid droplets. The fluorescent probe Lipi-Yellow of the present invention is a polarity-sensitive lipid droplet fluorescent probe with high photostability and staining selectivity, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : H NMR spectrum of the fluorescent probe Lipi-Yellow prepared in Example 1 of the present invention;

[0014] Figure 2 : Absorption and emission spectra of the fluorescent probe Lipi-Yellow prepared in Example 1 of the present invention measured in organic solvents toluene and dichloromethane; wherein the left part is the absorption spectrum and the right part is the emission spectrum;

[0015] Figure 3 :The co-localization photo of the fluorescent probe Lipi-Yellow and the lipid droplet fluorescent probe LDs-Red in HeLa cells; the cell nucleus was stained with Hoechst 33342;

[0016] Figure 4 : Quantification of the signal-to-noise ratio of the fluorescent probe Lipi-Yellow and lipid droplet fluorescent probes Nile Red and BODIPY in HeLa cells;

[0017] Figure 5 :Multicolor confocal imaging of the fluorescent probe Lipi-Yellow in living HeLa cells; revealing the distribution and activities of different organelles in one image;

[0018] Figure 6:The fluorescent probe Lipi-Yellow was used to monitor the polarity changes of intracellular lipid droplets in spectral scanning imaging, indicating that the maximum emission wavelength red-shifted by up to about 20nm after oleic acid stimulation for 24 hours; among them, the spectrum was fitted with a Gaussian function to obtain the emission maximum. DETAILED DESCRIPTION

[0019] Example 1: Synthesis of Lipi-Yellow

[0020] (1) 4,5-Diaminophthalonitrile (0.95 g, 5 mmol), 2-bromo-1-(4-bromophenyl)ethan-1-one (1.38 g, 6 mmol), water (25 mL) and hexadecyltrimethylammonium bromide (0.46 g, 1.25 mmol) were mixed and stirred at 100°C for 16 hours. After cooling, the mixture was poured into ice water and filtered to obtain a filter cake, which was washed with anhydrous ethanol to obtain intermediate 1 (1.7 g). The mixture was directly reacted in the next step without further purification.

[0021] (2) The intermediate product 1 (1 g) was mixed with diphenylamine (1.17 g, 6.6 mmol), tridibenzylideneacetone dipalladium (100 mg), cesium carbonate (7.9 g, 24 mmol), and tri-tert-butylphosphine (0.15 mmol), added to 80 mL of o-xylene, and refluxed at 140 ° C for 16 h. After cooling, it was poured into 200 mL of water and extracted three times with 500 mL of dichloromethane. The organic layers were combined, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane: petroleum ether volume ratio = 1:1) to obtain the final product Lipi-Yellow (0.65 g, yield 52%).

[0022] 1H NMR (400 MHz, DMSO) δ (TMS, ppm): 9.76 (1H, s), 8.93 (1H, s), 8.87 (1H, s), 8.31 (2H, d), 7.44 (4H, t), 7.23-7.18 (6H, m), 7.05 (2H, d), proving that the product of the target structure was prepared.

[0023] Example 2: Determination of the absorption-emission spectrum of the fluorescent probe Lipi-Yellow prepared in Example 1

[0024] The fluorescent probe Lipi-Yellow synthesized in Example 1 was prepared into a 10 μM solution with 10 mL toluene solvent. The absorption spectrum was obtained by scanning with a UV-visible spectrophotometer in the wavelength range of 300-700 nm, and the fluorescence emission spectrum was obtained by collecting with an optical fiber fluorescence spectrometer under 470 nm excitation. The data was processed with Origin software to obtain the absorption-emission spectrum of the fluorescent probe Lipi-Yellow in organic solvents toluene and dichloromethane (the left part is the absorption spectrum, and the right part is the emission spectrum), which illustrates the absorption-emission peak position of the fluorescent probe Lipi-Yellow and its large Stokes shift, as shown in FIG. Figure 2 shown.

[0025] Example 3: Subculture of HeLa cells

[0026] All percentages in this embodiment are volume percentages.

[0027] HeLa cell lines were cultured in an incubator at 37°C and 5% CO2 concentration, and the culture medium solution was high-glucose DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture). Among them, fetal bovine serum, double antibody and high-glucose DMEM were purchased directly from a biological reagent company.

[0028] The whole experiment was conducted on a clean bench. First, the reagents trypsin and culture medium solution required in the experiment were sterilized by high temperature using an alcohol lamp to prevent the cells from being contaminated. Then, the HeLa cells to be passaged were taken out of the incubator, the culture medium solution was sucked away with a pipette, trypsin was added, and it was slowly shaken two to three times to digest it. Next, the culture bottle was placed in the incubator and taken out after waiting for 2 minutes. After that, fresh culture medium solution was added to stop trypsin from continuing to digest. Start shaking slowly. If there are adherent cells that have not fallen off, use a pipette to blow until the cells are completely detached. Finally, some cells are sucked away and new culture medium solution is added. It is worth noting that before each cap is closed, the bottle mouth should be placed above the alcohol lamp to heat the fire, and the bottle body and bottle cap should be disinfected with alcohol spray to ensure that the entire process is sterile. The cells in the culture dish are used for confocal imaging experiments after the concentration is appropriate.

[0029] Example 4: Co-staining experiment of the fluorescent probe Lipi-Yellow prepared in Example 1 and the published lipid droplet fluorescent probe LDs-Red in HeLa cells

[0030] We cultured HeLa cells in a cell culture dish and placed them in a CO2 cell culture incubator for 2 days to make their growth morphology more mature and conducive to imaging observation. After taking them out of the incubator, we used a pipette to remove the original culture medium solution in the culture dish, and added DMEM+ containing probes (20μM nuclear dye Hoechst 33342, 2μM LDs-Red and 2μM Lipi-Yellow) and 1% DMSO in turn, and placed them in a CO2 incubator for staining for 2 hours. After that, the cells were repeatedly washed three times with HBSS, and finally 2mL HBSS was added for incubation and used for imaging. A complete cell morphology was determined by labeling the cell nucleus with Hoechst 33342, and then the cells were imaged in Figure 3 a and Figure 3 In b, we obtained the confocal images of Lipi-Yellow and LDs-Red stained lipid droplets; we continued to merge these two images to obtain Figure 3 c; The Pearson correlation coefficient R value of Lipi-Yellow and LDs-Red dyes was calculated to be 0.91 ( Figure 3 d), the results showed that Lipi-Yellow can specifically stain lipid droplets.

[0031] Example 5: Signal-to-noise ratio experiment of the fluorescent probe Lipi-Yellow prepared in Example 1 and the lipid droplet fluorescent probe Nile Red and BODIPY 493 / 503 in HeLa cells

[0032] We selected Lipi-Yellow, Nile Red and BODIPY 493 / 503 at a concentration of 2 μM to stain the cells for 2 h, and obtained the following results through confocal imaging: Figure 4 The imaging results are shown in a. We can clearly see that the Lipi-Yellow staining channel produces bright fluorescence signals at the lipid droplet marker position, while Nile Red and BODIPY 493 / 503 have strong background fluorescence signals. In order to further quantitatively compare the staining selectivity of the three fluorescent probes, we selected Figure 4 The area of ​​interest in a is enlarged to Figure 4 b. Figure 4 b and Figure 4As in a, from left to right, they represent three fluorescent probes: Lipi-Yellow, Nile Red, and BODIPY 493 / 503. In each cell, the fluorescence signal intensity at 10 different lipid droplet positions was selected and compared with the signal intensity in the adjacent cytoplasm to obtain the signal-to-noise ratio values ​​of lipid droplets stained by three different fluorescent probes. The data showed that the signal-to-noise ratio of Lipi-Yellow was as high as 67, while the signal-to-noise ratios of Nile Red and BODIPY 493 / 503 were around 17 and 24, respectively. Lipi-Yellow is more than three times that of Nile Red and BODIPY 493 / 503, and has superior lipid droplet staining selectivity (such as Figure 4 b gives the statistical results).

[0033] Example 6: Observation of the interaction between lipid droplets and organelles by multicolor imaging using the fluorescent probe Lipi-Yellow prepared in Example 1

[0034] Live HeLa cells were stained in DMEM+ containing the commercial mitochondrial dye MitoTracker Deep Red (50nM), the nuclear dye Hoechst 33342 (20μM) and 0.5% DMSO in a CO2 incubator for 20 minutes. After washing with fresh medium, the cells were further stained in DMEM+ containing the lysosomal dye LysoTracker Green (200nM), the lipid droplet fluorescent probe Lipi-Yellow (2μM) and 1% DMSO in a CO2 incubator for 40 minutes. Without washing, HeLa cells were imaged using a Leica TCS SP8 microscope, and the fluorescence signals of different channels were selectively excited and detected by line-by-line sequential scanning. The imaging results are shown in Figure 2. Figure 5 As shown in a. Figure 5 In a, we can clearly see the morphology of four organelles (nucleus, lipid droplets, lysosomes, and mitochondria); Figure 5 b shows a merged image of the four organelles, which allows the distribution status of the four organelles to be observed simultaneously in one image, providing a research basis for the subsequent dynamic monitoring of the interactions between the organelles.

[0035] Example 7: The fluorescent probe Lipi-Yellow prepared in Example 1 is used to monitor the polarity changes of intracellular lipid droplets in spectral scanning imaging

[0036] As a control group, we took the culture dish covered with HeLa cells in Example 3 out of the incubator, removed the original DMEM culture solution, added DMEM culture solution containing 2μM Lipi-EP and 1% DMSO, put it back into the incubator for incubation for 2 hours, and then took it out. Rinse it three times with fresh culture medium solution, and perform spectral scanning imaging; as an experimental group, after removing the original DMEM culture solution, add DMEM culture solution containing 400μM oleic acid (OA) and 1% DMSO, put it back into the incubator for incubation for 24 hours, and then take it out. Rinse it three times with fresh culture medium solution, and then add DMEM+ solution containing 2μM Lipi-Yellow and 1% DMSO, place it in a CO2 incubator for further staining for 2 hours, and perform spectral scanning imaging. First, Figure 6 In a, we can see the dual-channel imaging images of the cell nucleus and lipid droplets without OA stimulation, and the arrows indicate the location of the nuclear lipid droplets (nLDs) ROI1 and the locations of the cytoplasmic lipid droplets (cLDs) ROI 2 and ROI 3. Combined with the results of spectral scanning imaging, we obtained that the scanning wavelength corresponding to the location of the nuclear lipid droplets (nLDs) ROI1 is 568nm, and the scanning wavelengths corresponding to the locations of the cytoplasmic lipid droplets (cLDs) ROI 2 and ROI 3 are 569nm and 567nm respectively; Figure 6 In b, we obtained dual-channel imaging images of the nucleus and lipid droplets after oleic acid stimulation for 24 hours (OA-24h). The scanning wavelength corresponding to ROI1 at the position of nuclear lipid droplets (nLDs) changed to 584nm, and the scanning wavelengths corresponding to ROI 2 and ROI 3 at the position of cytoplasmic lipid droplets (cLDs) also changed to 586nm and 587nm. According to the comparison results between the control group and the experimental group, we found that the position of nuclear lipid droplets was 16nm red-shifted after oleic acid stimulation for 24 hours compared with the unstimulated state, and the position of cytoplasmic lipid droplets was also red-shifted by 17nm and 20nm, respectively. Therefore, we assume that the polarity of lipid droplets will increase after oleic acid stimulation.

Claims

1. A yellow light emitting fluorescent probe with polarity-sensitive properties, characterized in that: It is a donor-acceptor structure, and its structural formula is shown below, 2. Use of a yellow light emitting fluorescent probe with polarity-sensitive properties as described in claim 1 in the preparation of a cell lipid droplet imaging reagent.

3. The use of a yellow light emitting fluorescent probe with polarity sensitivity as claimed in claim 2 in the preparation of a cell lipid droplet imaging reagent, characterized in that: The cells are HeLa cells.

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