A fluorescent imaging probe for cell lipid droplets based on indolinone derivatives and its application in visualizing lipid droplets during cell ferroptosis

Abstract

A cell lipid droplet fluorescent imaging probe based on indolinone derivatives and its application, belonging to the field of biological imaging technology, the structural formula of the fluorescent probe is shown below. The present invention also discloses the application of the fluorescent probe in specifically labeling lipid droplets in HeLa cells and visualizing the changes in lipid droplet morphology, space and quantity during HeLa cell ferroptosis. Experiments have confirmed that the fluorescent probe Lipi-EP of the present invention is a lipid droplet fluorescent probe with ultra-high photostability and staining selectivity, and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging technology, and specifically relates to a cell lipid droplet fluorescent imaging probe based on indolinone derivatives and its application in specifically marking cell lipid droplets and visualizing the changes in lipid droplet morphology, space and quantity during cell ferroptosis. Background Art

[0002] Ferroptosis is a new cell death regulation form discovered in recent years that is dependent on iron and is different from traditional apoptosis, necrosis and autophagy. Recent studies have shown that ferroptosis is closely related to various physiological and pathological processes, such as cancer and neurodegenerative diseases, ischemic organ damage, T cell immunity, etc. Ferroptosis is a process that produces reactive oxygen species through the catalysis of the Fenton reaction, which further promotes lipid peroxidation, causes lipid peroxide accumulation, and inhibits the lipid repair enzyme glutathione peroxidase 4 (GPX4), thereby inducing ferroptosis. Lipid droplets (LDs) are the main organelles for storing neutral lipids, and therefore are inevitably related to the regulation of ferroptosis. Therefore, the study of lipid droplets during ferroptosis, especially the in situ revelation of the changes in lipid droplets during this process, can provide abundant and real-time information about lipid droplets, which is very important for studying the mechanism of ferroptosis formation.

[0003] It is well known that fluorescence imaging is a powerful method to monitor subcellular organelle behavior with the ability to detect in situ and in real time. So far, many lipid droplet fluorescent probes have been developed for lipid droplet imaging and dynamic tracking. In particular, some of these probes have been used to study the changes in lipid droplets during ferroptosis formation, greatly promoting the study of ferroptosis. However, these lipid droplet fluorescent probes are usually not suitable for long-term fluorescence imaging due to the limitations of insufficient photostability and / or relatively low specificity of lipid droplet staining. Therefore, monitoring the changes in ferroptotic lipid droplets in a long-term, in situ and real-time manner is a very important but challenging task. Summary of the invention

[0004] In view of the shortcomings of the existing cell lipid droplet imaging fluorescent probe technology, the problem to be solved by the present invention is to provide a cell lipid droplet fluorescent imaging probe based on indolinone derivatives and its application in specifically marking cell lipid droplets and visualizing the changes in lipid droplet morphology, space and quantity during cell ferroptosis.

[0005] The cell lipid droplet fluorescence imaging probe (Lipi-EP) based on indolinone derivatives described in the present invention has the following structural formula:

[0006]

[0007] Its preparation reaction formula is as follows:

[0008]

[0009] In view of the insufficient photostability and lipid droplet staining selectivity of existing cell lipid droplet fluorescence imaging probes, the present invention selected a fluorescent molecule based on indole derivatives in the development of a new lipid droplet imaging fluorescent probe. This fluorescent molecule exhibits high photostability and excellent lipid droplet staining selectivity. The present invention successfully performed time-lapse multicolor imaging and time-lapse three-dimensional imaging using this probe, and monitored the application of lipid droplet morphology, space and quantity changes in real time during ferroptosis.

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

[0011] The experimental results confirm that the fluorescent probe Lipi-EP described in the present invention has excellent photostability and staining selectivity. Under the same staining and imaging conditions, Lipi-EP exhibits better photostability and staining selectivity than commercial lipid droplet fluorescent probes BODIPY and Nile Red. Therefore, the fluorescent probe Lipi-EP can be used as a specific marker for lipid droplets, and can display the changes in lipid droplet morphology, space, and quantity during cell ferroptosis for a long time, in situ, and in real time. These fluorescence imaging results provide rich information about lipid droplets from multiple angles (morphology, space, and quantity), helping us to gain a deeper understanding of the changes in lipid droplets during ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Absorption-emission spectrum of the fluorescent probe Lipi-EP prepared in Example 1 of the present invention in toluene (the left dotted line part is the absorption spectrum, and the right solid line part is the emission spectrum); the absorption peak of Lipi-EP in toluene solution is at 470nm, and the emission peak is at 490nm.

[0013] Figure 2 : Cell survival bar graph of HeLa cells stained with different concentrations of the fluorescent probe Lipi-EP for 24 hours; the cell survival rates were all above 90%, proving the good biocompatibility of the fluorescent probe.

[0014] Figure 3 :Co-localization photos of fluorescent probe Lipi-EP and lipid droplet fluorescent probe LDs-Red in HeLa cells;

[0015] The cell nuclei were stained with Hoechst 33342.

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

[0017] Parts a and b of the figure are imaging pictures and local magnified pictures of HeLa cells stained with fluorescent probes Lipi-EP, BODIPY and Nile Red, respectively; part c of the figure is the fluorescence intensity ratio of the circled lipid droplet area to the circled cytoplasm area.

[0018] Figure 5 :Quantification of the photostability of the fluorescent probe Lipi-EP and lipid droplet fluorescent probes Bodipy and Nile Red in HeLa cells;

[0019] Part a of the figure shows the 1st, 10th and 50th photos of 50 consecutive images of the same area after HeLa cells were stained with fluorescent probe Lipi-EP, BODIPY and Nile Red; Part b of the figure shows the trend curve of the relative fluorescence intensity of these 50 photos as a function of the number of images. This figure shows that the probe Lipi-EP has good photostability.

[0020] Figure 6 : Photos and curves of the changes in lipid droplet size during ferroptosis monitored in real time by the fluorescent probe Lipi-EP in 360-minute time-lapse multicolor imaging, indicating that the size of lipid droplets remains basically unchanged during ferroptosis.

[0021] The horizontal axis is time, and the vertical axis is the diameter of the lipid droplet; the size information of the lipid droplet is obtained by counting the images at each time point.

[0022] Figure 7 :The fluorescent probe Lipi-EP monitors the changes in the number and distribution of lipid droplets in real time during ferroptosis in 360-minute time-lapse three-dimensional imaging. Photos and curves show that the number of lipid droplets decreases during ferroptosis

[0023] The horizontal axis is time, and the vertical axis is the number of lipid droplets. The change in the color of the lipid droplets means the different depths of the lipid droplets. The number of lipid droplets is obtained by the maximum projection of the three-dimensional image at each time point. DETAILED DESCRIPTION

[0024] Embodiment 1:

[0025] 1. Synthesis of Compound 2

[0026] According to the literature, dimethyl dihydroxyfumarate ( ED, Romanazzi, G., Yumusak, C., Coskun, H., Monkowius, U., Voss, G., Burian, M., Lechner, RT, Demitri, N., Redhammer, GJ, Sünger, N., Suranna, GP and Sariciftci, S., Adv. Funct. Mater., 25 (2015), 776-787). Concentrated hydrochloric acid (0.65 mL, 7.8 mmol) was added to a mixed solution of dimethyl dihydroxyfumarate (11.1 g, 63.0 mmol) and 4-(tert-butyl) aniline (20.8 g, 139 mmol) in methanol (50 mL) and stirred at reflux temperature for 5 h. The reaction mixture was then slowly cooled to room temperature and filtered through a sintered glass filter (G4). The collected solid was washed three times with cold methanol (150 mL) and then dried under vacuum to give 12.3 g (71%) of compound 2 as a white solid.

[0027] 1 H NMR (500MHz, DMSO): 7.57 (s, 2H), 7.15 (d, J = 8.5Hz, 4H), 6.64 (d, J = 8.5Hz, 4H), 3.63 (s, 6H), 1.21 (s, 18H). ESI-MS: 437.76 [MH] + (calcd:437.25).

[0028] 2. Synthesis of Compound 3

[0029] In a 1L round-bottom flask, a mixture of Dow's heat carrier A (260mL) and compound 2 (10.9g, 24.7mmol) was stirred vigorously and heated to 260 degrees Celsius to reflux within 30 minutes, and the reaction was continued for 30 minutes, and then slowly cooled to room temperature within 18 hours, and the precipitation of a brown solid product was observed. The solid was collected on a sintered glass filter (G4) and washed three times with petroleum ether (100mL). Finally, the solid was dried under vacuum to obtain 6.68g (66%) of yellow powder as compound 3.

[0030] 1 H NMR(500MHz,DMSO):11.74(s,1H),8.03(s,1H),7.83–7.77(m,2H),7.38(s,1H),7.13(d,J=8 .5Hz,2H),6.64(d,J=8.5Hz,2H),3.67(s,3H),1.34(s,9H),1.23(s,9H).ESI-MS:405.75[MH] +(calcd:406.53).

[0031] 3. Synthesis of compound 4

[0032] A mixture of 10 g of polyphosphoric acid and compound 3 (1.00 g, 2.46 mmol) was heated at 120°C for 2.5 h under nitrogen. The mixture was cooled to 50°C, and then water (100 ml) was slowly added, keeping the temperature constant until the exothermic process stopped. After cooling to room temperature, the precipitate was collected on a sintered glass filter (G4) and rinsed with water. The crude product was dried under vacuum to obtain compound 4, which was used in the next step without further purification.

[0033] 4. Synthesis of Lipi-EP

[0034] Sodium hydroxide solution (25.0 mM, 20 mL) and 1-bromopropane (3.7 mL, 50 mmol) were added to a mixture of compound 4 (0.745 g) and tetrabutylammonium bromide (120 mg, 0.372 mmol) in toluene (20 mL) and ethanol (2 mL), and refluxed under nitrogen for 48 hours. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with CH2Cl2. The organic layer was washed with saturated brine, then dried over anhydrous MgSO4, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 0.086 g (0.197 mmol) of green solid, which was the fluorescent probe Lipi-EP.

[0035] 1 H NMR(400MHz, CDCl3):8.43(d,J=2.3Hz,2H),7.87–7.82(m,2H),7.67(d,J=9.2 Hz,2H),4.81(t,4H),2.09–2.00(m,4H),1.46(s,18H),1.00(t,J=7.4Hz,6H). 13 C NMR(101MHz, CDCl3):174.92,145.12,140.08,131.87,125.18,122.46,116.32,51.33,34.67,31.26,23.41,11.25.ESI-MS:458.07[MH] + (calcd:457.29).

[0036] Example 2: Determination of the absorption-emission spectrum of the fluorescent probe Lipi-EP prepared in Example 1

[0037] The fluorescent probe Lipi-EP synthesized in Example 1 was prepared into a 10 μM solution using 10 mL of toluene solvent. The absorption spectrum was obtained by scanning with a UV-visible spectrophotometer in the wavelength range of 300 to 700 nm, and the fluorescence emission spectrum was obtained by collecting the fluorescence spectrum under excitation at 470 nm using a fiber optic fluorescence spectrometer. The data was processed using Origin software to obtain the following: Figure 1 The absorption-emission spectrum of the fluorescent probe Lipi-EP in toluene solution shown (the left dashed line part is the absorption spectrum, and the right solid line part is the emission spectrum) illustrates the absorption-emission peak position of the fluorescent probe Lipi-EP and its large Stokes shift.

[0038] Example 3: Cultivation of HeLa cells

[0039] All percentages in this embodiment are volume percentages.

[0040] HeLa cell lines were cultured in an incubator at 37°C and 5% CO2 concentration, and the culture medium 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.

[0041] When the cells grow to the logarithmic phase, we perform cell subculture treatment: after aspirating the original 5mL culture medium in the cell culture bottle, wash the cell surface with 2mL DMEM culture medium without fetal bovine serum, aspirate the culture medium and then digest the cells with 0.5mL trypsin for 2 minutes. After most of the cells are detached from the wall, add 2mL 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 bottle 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.

[0042] Example 4: Test of cytotoxicity of the fluorescent probe Lipi-EP prepared in Example 1

[0043] We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to test the cytotoxicity of the fluorescent probe Lipi-EP. HeLa cells were seeded in 96-well plates (1*10 4cells), 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 probe Lipi-EP 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 they were returned to the cell culture incubator for continued culture 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 value of each well was measured at 490 nm with a microplate reader. Because only living cells can react with the MTT reagent to generate formazan crystals, we can compare the average absorbance value of the wells with different concentrations in each group with the average absorbance value of the control group (10 wells with a probe concentration of 0) to calculate the cell survival rate. The results are as follows. Figure 2 As shown, it indicates that the fluorescent probe Lipi-EP has little cytotoxicity, and the fluorescent probe Lipi-EP at a concentration of 10.0 μM will not affect the normal growth of HeLa cells within 24 hours.

[0044] Example 5: Co-staining experiment of the fluorescent probe Lipi-EP prepared in Example 1 and the lipid droplet fluorescent probe LDs-Red in HeLa cells

[0045] We cultured HeLa cells in a 20 mm diameter glass-bottomed culture dish and propagated them in a CO2 incubator for 2 days. After taking it out of the incubator, remove the original DMEM culture medium in the culture dish, add 1 mL of DMEM culture medium containing Lipi-EP (500 nM), LDs-Red (2 μM) and 1% (volume fraction) DMSO, and continue to culture it in a cell culture incubator for 2 hours. After taking it out, wash it 3 times with HBSS solution, and then perform fluorescence imaging in HBSS solution. Figure 3 As shown, we can clearly observe that the fluorescent probe Lipi-EP prepared in Example 1 and the lipid droplet fluorescent probe LDs-Red can achieve good co-localization in HeLa cells, indicating that the fluorescent probe Lipi-EP prepared in Example 1 has excellent cell lipid droplet specificity.

[0046] Example 6: Photostability test of the fluorescent probe Lipi-EP prepared in Example 1

[0047] After we took out the two culture dishes filled with HeLa cells in Example 5 from the cell culture incubator, we removed the original DMEM culture medium, added DMEM culture medium (containing 1% DMSO) containing 2μM Lipi-DSB, 2μM BODIPY and 2μM Nile Red respectively, put them back into the incubator and continued to culture for 2 hours, then took out the three culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. We selected three areas in each of the three culture dishes, and then imaged 50 consecutive images respectively. We found that BODIPY and Nile Red would be photobleached quickly, while the relative fluorescence intensity of the fluorescent probe Lipi-EP prepared in Example 1 could still be maintained at 95% after 50 images, which shows its excellent photostability. Figure 5 shown.

[0048] Example 7: Observation of lipid droplets by multicolor time-lapse imaging of the fluorescent probe Lipi-EP prepared in Example 1 during ferroptosis

[0049] After we took the culture dish filled with HeLa cells in Example 5 out of the incubator, we removed the original DMEM culture solution, added DMEM culture solution containing 2 μM Lipi-EP and 1% DMSO, put it back into the incubator for 2 hours, and then took it out. Then we added DMEM culture solution containing 50 nM MitoTracker Deep Red, 20 μM Hoechst 33342 and 1% DMSO, put it back into the incubator for 20 minutes, and then took it out. We rinsed it with fresh culture medium solution three times, and then added DMEM culture solution containing 10 μM erastin, and performed in situ continuous confocal imaging, as shown in Figure 5. Figure 6 shown.

[0050] Example 8: Observation of lipid droplets by three-dimensional time-lapse imaging of the fluorescent probe Lipi-EP prepared in Example 1 during cell ferroptosis

[0051] After we took the culture dish filled with HeLa cells in Example 5 out of the incubator, we removed the original DMEM culture solution, added DMEM culture solution containing 2 μM Lipi-EP and 1% DMSO, put it back into the incubator and cultured for 2 hours before taking it out. We rinsed it three times with fresh culture medium solution, then added DMEM culture solution containing 10 μM erastin, and performed in situ continuous confocal three-dimensional imaging. We used Leica LAS X software to reconstruct the three-dimensional confocal images, as shown in Figure 2. Figure 7 shown.

Claims

1. A cell lipid droplet fluorescence imaging probe based on indolinone derivatives, the structural formula of which is shown below:

2. Use of the cell lipid droplet fluorescent imaging probe based on indolinone derivatives as described in claim 1 in the preparation of a reagent for specifically labeling lipid droplets in HeLa cells.

3. Use of a cell lipid droplet fluorescent imaging probe based on an indolinone derivative as described in claim 1 in the preparation of a reagent for visualizing changes in lipid droplet morphology, space and quantity during HeLa cell ferroptosis.

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