CN111057389B

CN111057389B - Fluorescent dye for specifically targeting intracellular lipid droplets and preparation method and application thereof

Info

Publication number: CN111057389B
Application number: CN201911298514.9A
Authority: CN
Inventors: 王振洋; 杨林林; 张忠平; 刘变化; 韩光梅; 赵君
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2021-08-17
Anticipated expiration: 2039-12-17
Also published as: CN111057389A

Abstract

The invention discloses a fluorescent dye for specifically targeting intracellular lipid droplets, a preparation method and application thereof, wherein the fluorescent dye for specifically targeting intracellular lipid droplets takes 5- (dimethylamino) naphthalene-1-sulfonic acid as a fluorescent parent, and different types of amine or alcohol compounds are introduced into the parent molecular structure, and the structure is shown as follows:

wherein R is selected from one of the following groups:

R₁is oxygen or carbon, R₂And R₃Is hydrogen, C1-8 alkyl or phenyl. The fluorescent dye can specifically target lipid droplets in cells, and has good light stability and low cytotoxicity. In addition, the fluorescent dye is not only a universal lipid drop dye, but also can trace the movement state of lipid drops in cells in real time and distinguish the number and the size of the lipid drops in different cell linesProvides possibility for researching the relevance between the lipid drop and other organelles, and has wide application prospect in the biological field.

Description

Fluorescent dye for specifically targeting intracellular lipid droplets and preparation method and application thereof

Technical Field

The invention belongs to the field of synthesis and technical application, and particularly relates to a fluorescent dye for specifically targeting intracellular lipid droplets, and a preparation method and application thereof.

Background

Lipid droplets are intracellular, highly mobile organelles produced by the endoplasmic reticulum, which not only play a role in lipid storage during cellular metabolism, but also are involved in a variety of physiological processes, including membrane transport, membrane and lipoprotein formation, and intracellular signal transduction, etc. The lipid drop is formed by wrapping a phospholipid monomolecular layer with a plurality of functional proteins embedded on the surface, and contains central lipid-triglyceride and a small amount of cholesterol ester. Although the compositions of lipid droplets in different cells are similar, the number and the size of the lipid droplets are obviously different, and the differences not only can reflect the metabolic state of the cells, but also can distinguish the cell types. Under normal conditions, cells strictly control the storage and consumption of fatty acids in lipid droplets, and maintain the normal operation and health of the living body. However, lipid droplets and their associated proteins, when dysfunctional, can lead to diseases such as obesity, diabetes and atherosclerosis. Therefore, the development of non-invasive, long-term fluorescent dyes for specific imaging of lipid droplets and real-time monitoring of intracellular lipid droplet formation, growth, fusion and fission is of great importance for further understanding of the physiological and pathological roles of lipid droplets.

Although transmission electron microscopy can clearly observe intracellular lipid droplets, it is difficult to track the dynamic changes of lipid droplets and their association with other organelles using invasive microscopy. However, the high time-space resolution fluorescence imaging technology provides an ideal solution for studying the dynamic process of lipid droplets in organisms and related physiological functions. Currently, a variety of fluorescent dyes for intracellular lipid droplet specific labeling have been developed, which become powerful tools for monitoring intracellular lipid droplet behavior. However, most lipid droplet fluorescent dyes still have great disadvantages: 1) the dye synthesis process is complex, the purification is difficult, the time and the labor are wasted, the cost is high and the like; 2) the self-absorption phenomenon of the dye is easily caused by small Stokes displacement; 3) the cell is in a stress state due to great cytotoxicity, and the tracing of the physiological process of lipid droplets in a natural state is difficult to realize. In order to realize specific imaging and real-time dynamic monitoring of molecular events involving lipid droplets in complex organisms, the development of lipid droplet localization dyes with large stokes shift, low cytotoxicity and light stability is urgently needed. The development and construction of the fluorescent dye with excellent performance and specific targeting of intracellular lipid droplets can play a great promoting role in exploring physiological functions of the intracellular lipid droplets and association systems between the intracellular lipid droplets and other organelles.

Disclosure of Invention

The invention provides a fluorescent dye specifically targeting intracellular lipid droplets and a preparation method and application thereof, aiming at the technical problems in the existing lipid droplet dye. The fluorescent dye has the advantages of large Stokes shift, low fluorescence background and good biocompatibility, can specifically target lipid droplets in cells, can realize long-time observation of the movement process of the lipid droplets in the cells, and can specifically identify A549 cells from three cells, namely HeLa, A549 cells and HFF-1 cells. The probe is used for cell staining without cleaning and can be directly used for laser confocal imaging.

The fluorescent dye specifically targeting intracellular lipid droplets is prepared by taking 5- (dimethylamino) naphthalene-1-sulfonic acid (DNS) as a fluorescent parent substance, and introducing different types of amine or alcohol compounds into the parent molecular structure, wherein the structure of the fluorescent dye is shown as follows:

wherein R is selected from one of the following groups:

R₁is oxygen or carbon, R₂、R₃Each independently selected from hydrogen, C1-8 alkyl or phenyl.

The preparation method of the fluorescent dye specifically targeting intracellular lipid droplets comprises the following steps:

dissolving amine or alcohol compound and alkali in 10-100mL of organic solvent, stirring and reacting for 10-90min at room temperature, then slowly dripping 0.4-30mL of solution dissolved with compound DNS-Cl into the reaction solution, and continuously stirring and reacting for 1-24h at room temperature; and (4) carrying out suction filtration to remove insoluble alkali, removing the organic solvent by using a vacuum rotary evaporator, and purifying the crude product to obtain the target product.

The structural formula of the compound DNS-Cl is as follows:

the amine or alcohol compound is one of the following structural compounds:

wherein R is₁Is oxygen or carbon, R₂、R₃Each independently selected from hydrogen, C1-8 alkyl or phenyl.

The alkali is potassium carbonate, triethylamine or 4-N, N-dimethylpyridine and the like.

The molar ratio of the compound DNS-Cl to the amine or alcohol compound to the alkali is 0.5:1:1-1:3: 3.

The organic solvent is dichloromethane or anhydrous acetonitrile.

The purification is carried out by adopting a silica gel column chromatography separation method, and an eluent is formed by mixing dichloromethane and methanol according to the volume ratio of 10:1-150: 1.

The fluorescent dye specifically targeting intracellular lipid droplets is used as a detection reagent when the intracellular lipid droplets are specifically marked, and can trace the movement behavior of the lipid droplets in the cells in real time; the cells comprise adenocarcinoma human alveolar basal epithelial cells A549 and human normal mammary epithelial cells MCF 10A.

The fluorescent dye specifically targeting intracellular lipid droplets is used as a fluorescent probe for carrying out fluorescence imaging on lipid droplets in various cells and can distinguish the sizes and the number of different intracellular lipid droplets; the cells comprise A549 cells, human normal mammary epithelial cells MCF10A, mouse mononuclear macrophages RAW 264.7, human normal liver cells QSG 7701 and cervical cancer cells HeLa.

The fluorescent dye specifically targeting intracellular lipid droplets is used as a detection reagent to specifically identify A549 cells in three mixed cells of cervical cancer cells (HeLa), human skin fibroblasts (HFF-1) and adenocarcinoma human alveolar basal epithelial cells (A549).

The invention designs and synthesizes the lipid drop targeted fluorescent dye with low fluorescent background, large Stokes shift and good biocompatibility. The fluorescent dye has proper lipid solubility and low cytotoxicity, and can rapidly cross cell membranes to reach lipid droplets. The fluorescent dye can specifically mark lipid droplets in cells and is a universal lipid droplet positioning dye through a co-localization experiment with a commercial lipid droplet dye Nile Red. Compared with a commercial dye Nile Red, the Stokes shift of the dye can reach more than 210nm, and the good light stability and the specificity of lipid droplet marking enable the dye to be suitable for observing the dynamic change process of lipid droplets in real time. In addition, the fluorescent dye can specifically identify A549 cells by mixing three cells of HeLa, HFF-1 and A549 cells. At present, the preparation method and the application of the fluorescent dye for labeling intracellular lipid drops based on dansyl and amino or alcohol are not reported.

Compared with the prior art, the invention has the beneficial effects that:

1. the fluorescent dye synthesized by the invention is obtained by sulfonylation reaction, the reaction is a one-step synthesis method, the used materials are cheap, the synthesis is simple, the reaction condition is mild, and the purification is easy.

2. The ammonia or alcohol compound in the structure of the fluorescent dye synthesized by the invention can enable the probe to have proper lipophilicity, so that the probe can rapidly cross cell membranes and specifically mark lipid droplets in cells.

3. The fluorescent dye synthesized by the invention has good light resistance, can mark lipid drops for a long time, and can observe the movement of the lipid drops in living cells in real time.

4. The fluorescent dye synthesized by the invention is successfully used for specifically identifying A549 cells in three mixed cells of HeLa, HFF-1 and A549 cells.

5. The fluorescent dye has the advantages of large Stokes shift, good light stability and small cytotoxicity, and lays a foundation for monitoring the physiological activity of fat drops in cells in real time.

Drawings

FIG. 1 shows the UV-VIS absorption spectrum and fluorescence spectrum of the fluorescent dye DNS-M of the present invention in solvents with different polarities.

FIG. 2 shows the co-localization of the fluorescent dye DNS-M of the present invention with different commercial organelle dyes in A549 cells, respectively: such as the cell membrane dye CellTracker Deep Red, the lysosomal dye LysoTracker Deep Red, the mitochondrial dye MitoTracker Deep Red and the lipid droplet dye Nile Red. And respectively adding the commercial organelle fluorescent dye into different dish cells for incubation for 30min, replacing a fresh culture medium, adding DNS-M for continuous culture for 30min, and then carrying out confocal imaging. Green channel (DNS-M): the excitation wavelength is 405nm, and the collected emission peak range is 499-678 nm; red channel (lipid drop dye): excitation wavelength 543nm, and collection emission peak range is 548-680 nm; red channel (mitochondrial, lysosomal and cellular dyes): the excitation wavelength is 633nm, and the collection emission peak range is 638-747 nm. A scale: 10 μm.

FIG. 3 is a diagram showing the real-time tracing of the movement track of lipid droplets in cells by the fluorescent dye DNS-M of the present invention. The fluorescence images of the cells collected at different time points are respectively represented by different pseudo-colors, and then the fluorescence images collected at the close time points are superposed to observe the real-time movement of lipid drops in the cells. The excitation wavelength was 405nm, and the collected emission peak wavelengths were respectively 499-678 nm. The scale bar is 10 μm.

FIG. 4 shows the photostability test of the fluorescent dye DNS-M of the present invention. Then DNS-M is added into the cells for incubation for 30min, and then confocal fluorescence imaging is carried out. The cells are imaged for a plurality of times, the average value of the fluorescence intensity of the same area of the cells in the fluorescence images with the scanning times of 1, 10, 20, 30, 40, 50 and 60 times is counted respectively, and a fluorescence change curve is made. The excitation wavelength was 405nm, and the collected emission peak wavelengths were respectively 499-678 nm. The scale bar is 10 μm.

FIG. 5 shows that the fluorescent dye DNS-M of the invention specifically recognizes A549 cells from three mixed cells of HeLa, HFF-1 and A549 cells. Firstly, the three cells are inoculated in the same confocal glass dish, after 24 hours of culture, a fresh culture medium without calf serum is replaced, then 10 mu M DNS-M is added, and the culture is carried out for 30min for confocal imaging. The excitation wavelength was 405nm and the collected emission peak wavelength ranged from 499-678 nm. The scale bar is 20 μm.

FIG. 6 shows the co-localization imaging of the fluorescent dyes DNS-M and Nile Red in different cells. Nile Red (1. mu.M) was added to A549 cells, MCF10A, Raw 264.7.7 cells, QSG 7701 cells and HeLa cells, respectively, and after incubation for 30min, DMEM was replaced with fresh DMEM, DNS-M (10. mu.M) was added thereto, and after further incubation for 30min, confocal fluorescence imaging was performed. Green channel (DNS-M): excitation light wavelength 405nm, collected emission peak range: 499-678 nm; red channel (Nile Red): the wavelength of the excitation light is 543nm, and the collected emission peak range is 548-680 nm. The scale bar is 10 μm.

Detailed Description

Example 1: synthesis of the dye dansyl-morpholine (DNS-M)

81.0mg of morpholine (0.927mmol) and 128.1mg of anhydrous potassium carbonate (0.927mmol) were dissolved in 10mL of anhydrous acetonitrile and stirred at room temperature for 30 min. A10 mL anhydrous acetonitrile solution containing 250mg dansyl chloride (0.927mmol) was added dropwise to the above reaction system, and the reaction was continued with stirring at room temperature for 3 hours. After the reaction is finished, potassium carbonate powder is removed by suction filtration, and a vacuum rotary evaporator is usedThe acetonitrile was removed. Purifying the obtained green powder by column chromatography to obtain the target product DNS-M, wherein the eluent is CH₂Cl₂/CH₃OH(v/v＝70:1)。¹H NMR(400MHz,CDCl₃):δ8.58(d,1H),8.41(d,1H),8.21(dd,1H),7.54(m,2H),7.20(d,1H),3.69(t,4H),2.89(s,6H).¹³C NMR(100MHz,CDCl₃):δ151.8,132.3,130.9,130.8,130.5,130.1,128.1,123.2,119.7,115.4,66.3,45.4,45.4.HR-MS(m/z,ESI)calculated for C₁₆H₂₀N₂O₃S:[M+Na]⁺＝343.1092,found 343.1073；[2M+Na]⁺＝663.2287,found 663.2296.

Example 2: UV-VISIBLE ABSORPTION SPECTRUM AND FLUORESCENCE SPECTRUM OF DYE DNS-M IN SOLVENTS WITH DIFFERENT POLARITY

DNS-M was dissolved in ethanol and prepared as a stock solution at a concentration of 10 mM. mu.L of the stock solution having a DNS-M concentration of 10mM was taken out from the stock solution, added to 2mL of dichloromethane, chloroform, ethyl acetate, acetonitrile, ethanol, DMF and water, and the UV-visible absorption and fluorescence spectrum curves of the fluorescent dye in the above solvent were measured. As shown in FIG. 1A, the absorption peak and the absorbance of the dye DNS-M in organic solvent are basically the same, and only the absorption peak of the dye in aqueous solution is blue-shifted by about 15nm, which shows that the polarity of the solvent does not influence the absorption of the dye basically. However, the polarity of the solvent not only has a very obvious influence on the fluorescence intensity of the dye, but also causes the dye to generate a significant red shift of 45nm in peak wavelength. In addition, the fluorescence intensity becomes significantly weaker with the increase in the polarity of the solvent, and only weak fluorescence is observed in water. However, since the substance contained within the lipid droplets is predominantly neutral lipids, the above spectral changes contribute to an improved signal-to-noise ratio of the dye in cellular lipid droplet imaging.

Example 3: co-localization of the dye DNS-M with different commercial organelle dyes in A549 cells

Commercial lysosomal dye LysoTracker Deep Red (1. mu.M), mitochondrial dye MitoTracker Deep Red (0.3. mu.M), cell membrane dye CellTracker Deep Red (1. mu.M) and lipid drop dye Nile Red (1. mu.M) were added to A549 cells in different dishes, respectively, at 37 ℃ with 5% CO₂Culturing under the conditions 3After 0min, the medium was replaced with fresh medium. Then, the fluorescent dye DNS-M prepared in example 1 was added to the above-mentioned cells to a final concentration of 10. mu.M, and after incubation for 30min, laser confocal imaging was performed, and the imaging results are shown in FIG. 2. As can be seen from the figure, the green spot from the dye DNS-M almost does not overlap with the red spot from the lysosomal, mitochondrial and cell membrane dyes, indicating that the dye DNS-M does not have lysosomal, mitochondrial or cell membrane localization capability. While the green spot of the dye DNS-M almost completely overlaps with Nile Red from the lipid droplet dye, suggesting that the dye DNS-M can specifically target intracellular lipid droplets. Although the dye contains morpholine which is a commonly used targeting group of lysosome, the dye DNS-M does not mark lysosome but specifically marks lipid droplets, probably because sulfonic acid groups in parent molecules of the dye are directly connected with secondary amines in morpholine, the alkalinity of the amines is weakened, the lipid solubility of the amines is improved, and the dye is more prone to accumulate lipid droplets in cells rather than lysosomes.

Example 4: photostability of the dye DNS-M

Culturing HeLa cells in a glass dish special for laser confocal imaging for 24h, replacing a serum-free fresh culture medium, adding 10 mu M of dye DNS-M into the cells, continuously culturing for 30min, and directly performing laser confocal fluorescence imaging. Cells in the same field were imaged a plurality of times, and the mean values of the fluorescence intensities at the fixed sites of the fluorograms were counted for 1, 10, 20, 30, 40, 50, and 60 imaging times, and then the change curves of the mean fluorescence intensities were plotted. As can be seen from FIG. 3A, the fluorescent light remained bright in the cell after the probe was subjected to 60 consecutive imaging, indicating that the probe has good light stability. The change of the fluorescence intensity of the dye in the cell after different times of scanning can be visually seen from the change curve of the fluorescence intensity (figure 3B), and the fluorescence intensity of the probe is basically kept stable after the cell is imaged for 60 times, which shows that the probe has very good light stability and is suitable for observing the change of lipid drops in the cell for a long time.

Example 5: dye DNS-M traces the movement track of lipid drop in cell in real time

Respectively inoculating the tumor cell A549 and the normal cell MCF10A in different special laser confocal glass dishes, culturing for 24h, replacing a fresh culture medium, adding 10 mu M of dye DNS-M into the cells, and directly performing laser confocal fluorescence imaging after culturing for 30 min. Selecting cells with proper lipid droplets, collecting fluorescence images of the cells at different time points, wherein the selected time is 0min, 2min, 4min and 6min, imaging the cells by adopting different pseudo colors, superposing the fluorescence images at the close time points by using software ImageJ, and observing the movement track of the lipid droplets in the cells in real time by comparing fluorescence spots with different colors. As shown in FIG. 4A, in the cell overlay at 0+2min, 2+4min and 4+6min, we found that the differently pseudo-colored fluorescent spots were substantially misaligned, indicating that very significant movement of lipid droplets occurred in the A549 cells. In contrast, in the fluorescence overlay of MCF10A cells (shown in FIG. 4B), the vast majority of the differently pseudocolored fluorescence spots were superimposed, indicating that the displacement and velocity of lipid droplet movement in MCF10A was relatively slow. The enlarged views in the lower right hand corner of FIGS. 4A and 4B show in particular the movement trajectories of lipid droplets in both cells. The results show that the lipid droplet is a fast-rotating organelle, and different cell types, the metabolic rate of cells, the pathological change degree and the like can all cause the remarkable difference of the motion behavior of the lipid droplet.

Example 6: specifically recognizing A549 cells in three mixed cells of HeLa, HFF-1 and A549 cells

HeLa + A549 cells, HFF-1+ A549 cells and HeLa + HFF-1+ A549 cells are respectively and uniformly mixed and then inoculated into different special laser confocal glass dishes, after 24 hours of culture, 10 mu M of dye DNS-M is respectively added into the cells, after 30 minutes of continuous culture, laser confocal fluorescence imaging is directly carried out, and the imaging result is shown in figure 5. As shown in the figure, the probe showed only very weak fluorescence in HeLa and HFF-1 cells, indicating that the lipid droplets were small in size and small in number in both cells. In contrast, the probe exhibited a bright and large spot of green fluorescence at the edge of a549 cells, indicating that more lipid droplets were distributed at the edge of a549 cells and the volume of the lipid droplets was relatively large. Among the three mixed cells, the probe was able to light only a549 cells, indicating that the probe was able to specifically recognize a549 cells among the above three cells (shown in the red dotted line box).

Example 8: co-localization of the dye DNS-M with the lipid drop dye Nile Red in different cells

Respectively inoculating A549 cells, MCF10A, Raw 264.7.7 cells, QSG 7701 cells and HeLa cells into a confocal special dish for culturing for 24h, adding 1 mu M Nile Red for culturing for 30min, replacing a fresh culture medium, adding 10 mu M dye DNS-M for continuously culturing for 30min, and carrying out confocal imaging. As shown in FIG. 6, the green fluorescence from the dye DNS-M and the Red fluorescence from Nile Red overlap well in all cells, indicating that the dye DNS-M is a universal lipid drop dye. Furthermore, there were significant differences in the brightness, size and number of fluorescent spots in different cells from the dye, indicating that the number and size of lipid droplets varied in different cell types. For example, the number of lipid droplets in a549 and MCF10A cells is large, whereas the number of lipid droplets in GSQ 7701 and Raw 264.7 cells is small, but the volume of lipid droplets is large. The tiny weak fluorescent spots in HeLa cells indicate that the volumes of lipid droplets in the cells are small. The degree to which the green spot from DNS-M overlaps the Red spot from Nile Red also reflects the rate of movement of lipid droplets in different cells. The probability of green and red spot overlap was significantly higher in MCF10A, Raw 264.7 and QSG 7701 cells than in a549 cells, indicating that the rate of lipid droplet movement in a549 cells was greater than that in other cells.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto. Numerous and varied simple alterations and substitutions will occur to those skilled in the art without departing from the spirit of the invention and the scope of the invention is to be determined from the appended claims.

Claims

1. Use of a fluorescent dye specifically targeting intracellular lipid droplets, wherein:

the fluorescent dye is used for preparing a detection reagent for specifically labeling lipid drops in cells, and the detection reagent can trace the movement behavior of the lipid drops in the cells in real time;

the structure of the fluorescent dye is shown as follows:

2. use according to claim 1, characterized in that:

the cells include adenocarcinoma human alveolar basal epithelial cells and human normal mammary epithelial cells.

3. Use of a fluorescent dye specifically targeting intracellular lipid droplets, wherein:

the fluorescent dye is used for preparing a fluorescent probe, and the fluorescent probe performs fluorescence imaging on lipid droplets in various cells and can distinguish the sizes and the number of the lipid droplets in different cells;

the structure of the fluorescent dye is shown as follows:

4. use according to claim 3, characterized in that:

the cells comprise adenocarcinoma human alveolar basal epithelial cells A549, human normal mammary epithelial cells MCF10A, mouse mononuclear macrophages RAW 264.7, human normal liver cells QSG 7701 and cervical cancer cells HeLa.

5. Use of a fluorescent dye specifically targeting intracellular lipid droplets, wherein:

the fluorescent dye is used for preparing a detection reagent for specifically identifying A549 cells in three mixed cells of HeLa, human skin fibroblast HFF-1 and A549 cells;

the structure of the fluorescent dye is shown as follows: