CN110981842A

CN110981842A - Fluorescent probe for distinguishing normal cells and cancer cells and specifically detecting lipid droplets and application

Info

Publication number: CN110981842A
Application number: CN201911118078.2A
Authority: CN
Inventors: 李朝辉; 孙远强; 魏星; 王霞; 屈凌波
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-04-10
Anticipated expiration: 2039-11-15
Also published as: CN110981842B

Abstract

The invention provides a fluorescent probe for specific detection of lipid droplets for distinguishing normal cells and cancer cells and its application, and its chemical structural formula is:

The fluorescent probe designed in the present invention has high selectivity for intracellular lipid droplets, can specifically mark the shape of lipid droplets, show distribution and application in living cells, tissues or living bodies, and can distinguish normal cells and cancer cells by fluorescence cells, normal tissue and tumor tissue.

Description

Fluorescent probe for distinguishing normal cells and cancer cells and specifically detecting lipid droplets and application

Technical Field

The invention relates to the technical field of organic small molecule fluorescent probes and biosensing, in particular to a fluorescent probe for specifically detecting lipid droplets and distinguishing normal cells and cancer cells and application thereof.

Background

Lipid Droplets (LDs) are of increasing interest due to their specific structure and physiological function. LDs are present widely in eukaryotic cells as lipid-rich subcellular organelles. LDs consist of a single layer of phospholipids with a neutral lipid core through which the relevant proteins are mounted. In addition to serving as energy stores, LDs are also involved in various physiological processes including increasing cell activation, migraine, apoptosis, and the like. There is increasing evidence that abnormalities in LDs are associated with the development of cancer. Therefore, the study of advantageous tumor markers is of particular importance for basic and clinical studies.

Lipid droplets have a large neutral core without water, so some lipophilic probes have been developed and used to image lipid droplets according to the "similar compatibility" principle. Among the more widely used are two commercial lipid droplet probes: nile Red and BODIPY. Nile Red is a lipophilic probe that, although it preferentially aggregates in lipid droplets, stains most structures inside the cell and also fluoresces weakly in water, thus causing a significant background noise, resulting in poor selectivity. To increase selectivity, another lipophilic probe, BODIPY, and other lipophilic lipid droplet probes were also used sequentially to image lipid droplets. Compared with Nile Red, the selectivity of the lipid droplets is improved, but the research requirements cannot be met, and further research on the lipid droplets is hindered. It follows that it is difficult to design lipophilic lipid droplet probes that rely solely on the lipophilicity of the lipid droplets for the purpose of highly selective imaging of lipid droplets.

Although many LDs fluorescent probes have been developed, most of them are limited to LDs themselves, and it is only rarely reported that normal organs and tumor organs are detected by intracellular polarity and lipid droplet differences, and solving this problem still has certain challenges. A novel fluorescent probe, which can realize the purpose of cancer diagnosis by sensing the change of the quantity and polarity of LD, is urgently needed not only at the cellular level but also in organs and living bodies.

Disclosure of Invention

The invention provides a fluorescent probe for distinguishing normal cells and cancer cells and specifically detecting lipid droplets and application thereof, and aims to solve the problem that coumarin is used as a matrix, provide a lipid droplet fluorescent probe with ultrahigh selectivity and application thereof in specifically marking or displaying the lipid droplet shape and distribution in living cells or tissues so as to realize imaging of lipid droplets in cancer cells and tumor tissues without background noise. Cancer diagnosis can be achieved by imaging changes in the number and polarity of LDs, and can be applied to cells, tissues and living bodies.

The technical scheme for realizing the invention is as follows:

a fluorescent probe for distinguishing normal cells from cancer cells and specifically detecting lipid droplets, wherein the chemical names of the fluorescent probe are 7- (dimethylamino) -2-oxo-2H-methylene-4-carboxylic acid ethyl ester and 7- (diethylamino) -2-oxo-2H-methylene-4-carboxylic acid ethyl ester, and the structural formula is as follows:

the preparation method of the fluorescent probe for distinguishing the normal cells from the cancer cells and specifically detecting the lipid droplets comprises the following steps: placing diethyl oxaloacetate and 3- (dimethylamino) phenol or 3- (diethylamino) phenol in a round-bottom flask, stirring for 3h at 120 ℃, cooling, extracting with dichloromethane and water, and purifying by a silica gel chromatographic column to obtain the fluorescent probe with a structure a or b.

The mass ratio of diethyl oxaminate to 3- (dimethylamino) phenol or 3- (diethylamino) phenol was 1: 1.

The fluorescent probe is applied to the specific labeling of lipid droplet form.

The fluorescent probes are capable of detecting lipid droplets in living cells, organs and living organisms, and for detecting lipid droplet polarity in living systems.

The fluorescent probe can image lipid drops in cancer cells and tumor tissues without background noise, and distinguish normal cells from cancer cells, normal tissues from tumor tissues by imaging the change of the number and polarity of LDs.

The specific content of the application comprises the following steps:

and respectively testing the ultraviolet visible absorption spectrum and the fluorescence spectrum of the probe storage solution added into the PBS buffer solution, wherein the excitation wavelength of the fluorescence is 420nm, and observing the change of the ultraviolet visible absorption spectrum and the fluorescence spectrum.

The change in fluorescence spectrum was: when the fluorescent material is excited by 420nm light, the fluorescence at 600nm is rapidly enhanced, and a response platform is reached in about 2 min.

The changes of the fluorescence imaging map are: incubating cells by using probe mother liquor, and collecting a fluorescence photo obtained by using a probe a under 405nm laser irradiation at the wavelength of 450-590nm by using a confocal microscope; the voltage obtained by Nile red under 488nm laser irradiation at the collection wavelength of 590-620nm is 20V, and the bright field transmission light detector is 360. Performing confocal imaging by using HePG-2, A549 cells and MCF-7 cells; confocal imaging was performed with 7702 cells, 3T3 cells under the same conditions.

The application comprises the following specific steps:

(1) PBS (10mM) buffer solution with pH 7.4 is prepared; the probe was weighed, dissolved in DMSO, and a 2mM probe stock solution was prepared accurately.

(2) After 2mL of PBS buffer solution was added to the cuvette, 4. mu.L of 2mM probe stock solution was added to react with different polar solutions, excitation was performed at 420nm, and collection wavelength was 400-.

(3) After 2mL of PBS buffer solution was added to the cuvette, 4. mu.L of 2mM probe stock solution was added to react with 1, 4-dioxane solution at different ratios, excitation was performed at 420nm, and the wavelength was collected at 400-700 nm.

(4) To the cuvette, 2mL of buffer solution at different pHs was added, and 4. mu.L of probe stock was added. Through analysis, the probe has a good linear relation in a physiological range, the detection limit is 0.00228 mu M, and the stability is high.

(5) And carrying out fluorescence imaging on the cells incubated with the fluorescent probes by using a confocal microscope.

(6) And carrying out fluorescence imaging on the living body incubated with the fluorescent probe by using a small animal imager.

The reaction formula for preparing the ultra-high selectivity lipid drop fluorescent probe is as follows:

the neutral core inside the lipid droplet is coated with a monolayer of amphiphilic phospholipid and in order to store the lipid droplet efficiently while maintaining the stability of the lipid droplet structure, the water inside the lipid droplet is drained off, so that the lipid droplet actually forms a unique amphiphilic structure comprising a polar head and an anhydrous interior. Based on such recognition, we have selected a coumarin which follows the ICT (intramolecular charge transfer) mechanism as the fluorescent parent, has strong lipophilicity, can have strong binding force with the neutral nucleus of lipid droplet, can realize specific targeting of lipid droplet, and is favorable for imaging without background noise. The luminescent property of the fluorophore is influenced by the polarity of the environment, namely the fluorescence intensity of the fluorophore in a low-polarity environment is obviously higher than that of the fluorophore in a high-polarity environment, so that the lipophilic fluorophore can emit strong fluorescence after targeting the interior of the low polarity of the lipid droplet, the effect of fluorescence switching is realized, and further, imaging without background noise is facilitated. The invention aims to solve the problem that coumarin is used as a matrix, and provides a lipid drop fluorescent probe with ultrahigh selectivity and application thereof in specifically marking or displaying the lipid drop shape and distribution in living cells or tissues or living bodies, which can specifically identify lipid drops in cancer cells so as to realize imaging of lipid drops in cancer cells and tumor tissues without background noise.

The living cells are preferably HepG-2 cells, 7702 cells, A549 cells, MCF-7 cells and 3T3 cells, and the tissues are preferably mouse muscle tissues, liver tissues and tumor tissues.

The invention has the beneficial effects that:

(1) the present invention provides a fluorescent probe with ultra-high selectivity that can be used to image lipid droplets without background noise. Experimental results prove that the method for detecting the polarity and the quantity of LDs in a life system can specifically target lipid droplets in cells and even more complex tissues, and the imaging effect of the method is far stronger than that of a commercialized lipid droplet probe Nile red. Meanwhile, the luminescent property of coumarin is not influenced by introducing an ester group part on the basis of coumarin, so that the fluorescent property of coumarin, namely ICT property, is kept by the probe, the fluorescence is influenced by the environmental polarity, the fluorescence intensity is reduced along with the increase of the environmental polarity, and meanwhile, the fluorescence peak position is in red shift. Therefore, when the probe is targeted to the inside of the lipid droplet with low polarity, the fluorescence intensity will be much greater than that in water, so that the fluorescence switching effect can be achieved. Meanwhile, the probe can be directly observed under a microscope without further washing after being dyed by the high signal-to-noise ratio and the low background noise. In addition, it stains very quickly (1 min), has very low cytotoxicity, and is compatible with other probes. Therefore, the probe can become a powerful tool for researching the lipid droplet and related activities, and the design concept can provide a universal guidance for the design of the probe of future membranous organelles including the lipid droplet.

(2) The high-selectivity lipid drop probe provided by the invention is used for detecting the LDs polarity in a living system, and can image lipid drops without background noise and identify used fluorescent probes of tumor cells, tissues and living bodies. Compared with other lipophilic lipid drop fluorescent probes with similar functions, the probe provided by the invention has the characteristics of ultrahigh selectivity, no background noise imaging, high dyeing capability, no washing, low cytotoxicity, capability of identifying tumor cells and the like.

In conclusion, the probe is a brand-new probe, has the characteristics of wide application range, high dyeing speed, low cytotoxicity and capability of specifically imaging lipid droplets in active cells, and has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows probe a¹H NMR spectrum.

FIG. 2 is a fluorescence spectrum of the action of probe a (4. mu.M) with different polar solvents in a PBS-buffered (10mM, pH 7.4) system.

FIG. 3 is a graph showing fluorescence spectra of probe a (4. mu.M) in PBS buffered (10mM, pH 7.4) with different ratios of 1,4 dioxane (DIOX/PBS), respectively.

FIG. 4 shows the viability of cells after treatment of HepG-2 with varying concentrations of probe a (0, 2, 4, 6, 8, 10, 15, 20. mu.M).

FIG. 5 is a diagram of a cell experiment study in which a probe recognizes lipid droplets.

FIG. 6 is a control and co-localization experiment of imaging of HepG-2 cells with Nile red (Nile red) and probe a.

FIG. 7 is a scatter plot of probe and Nie Red channel intensity.

FIG. 8 is a graph of time-dependent fluorescence imaging of probe a in mouse liver tissue, biopsy organs, and tumors stained with probe a (4.0. mu.M). Concentration: 10 mu M; λ ex ═ 430nm, λ em ═ 600 nm.

FIG. 9 is a photograph showing staining of a living body by probe a (4.0. mu.M), and imaging of a tumor using probe a in a tumor-bearing mouse xenograft model prepared by subcutaneous inoculation of HepG-2 cells. Mice were injected with a (50 μ M, 50mL) in the tumor area and normal area, respectively, and then imaged every 5 minutes.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

The structural formula of the probe a is as follows:

the preparation steps of the probe a are as follows: oxalyl oxalic acid diA mixture of ethyl ester (1.818g, 10mM) and 3- (dimethylamino) phenol (1.3718g, 10mM) was stirred at 120 ℃ for 3 hours. After cooling, the reaction mixture is washed with CH₂Cl₂Diluting and purifying by silica gel column Chromatography (CH)₂Cl₂) This gave an orange solid (1.07g, 45% yield).

Example 2

The structural formula of the probe b is as follows:

the preparation steps of the probe b are as follows: a mixture of diethyl oxaloacetate (1.818g, 10mM) and 3- (diethylamino) phenol (1.3718g, 10mM) was stirred at 120 ℃ for 3 hours. After cooling, the reaction mixture is washed with CH₂Cl₂Diluting and purifying by silica gel column Chromatography (CH)₂Cl₂) This gave an orange solid (1.07g, 45% yield).

Application of the probe a prepared in example 1:

1. change in fluorescence intensity of Probe a upon action with solvents of different polarity

Preparing a PBS (10mM) buffer solution with the pH value of 7.4; weighing probes, dissolving the probes by DMSO (dimethyl sulfoxide), and accurately preparing 2mM probe stock solution; after 2mL of 1, 4-dioxane, dichloromethane, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide, ethylene glycol, ethanol, acetonitrile, methanol and PBS buffer solution were added to each cuvette, 4. mu.L of a 2mM probe stock solution was added, and a fluorescence spectrum test was performed. As shown in FIG. 2, in the solutions with different polarities, the fluorescence intensities of the emission wavelengths of the probes have certain differences; 2mL of a solution containing 0-100% of DIOX (1, 4-Dioxane (DIOX)/PBS mixed at different ratios) was added to the cuvette, and 4. mu.L of a probe stock solution having a concentration of 2mM was added to perform fluorescence spectroscopy (see FIG. 3). In summary, it is shown that polarity has some effect on the probe.

2. Cytotoxicity assay of probes

Cell viability after HepG-2 treatment with varying concentrations (0, 2, 4, 6, 8, 10, 15, 20. mu.M) of probe a (FIG. 4) indicates that the probe is less cytotoxic.

3. Imaging picture of probe a on cancer cell (A549, MCF-7, HepG-2) and normal cell (7702, 3T3)

(a1) Is a cell image of A549 cells added with a probe a (4.0 mu M) in a fluorescence field; (a2) is a cell imaging graph of A549 cells added with a probe a (4.0 mu M) in an overlay field; (b1) is a cell imaging graph of MCF-7 cells added with a probe a (4.0 mu M) in a fluorescence field; (b2) is a cell imaging graph after adding a probe a (4.0 mu M) into MCF-7 cells in an overlay field; (c1) is a cell imaging picture of adding a probe a (4.0 mu M) into HepG-2 cells in a fluorescence field; (c2) is a cell imaging graph after adding a probe a (4.0 mu M) into HepG-2 cells in an overlay field; (d1) is a cell image of 7702 cells added with a probe a (4.0 mu M) in a fluorescence field; (d2) is an image of cells after 7702 cells are added with a probe a (4.0 mu M) in an overlay field; (e1) is a cell imaging graph of 3T3 cells added with a probe a (4.0 mu M) in a fluorescence field; (e2) is an image of 3T3 cells added with probe a (4.0. mu.M) in a superimposed field (as shown in FIG. 5); the excitation wavelength of 405nm is selected, and the wavelengths of 450 nm and 590nm are collected. Scale bar: 25 μm.

4. Control and co-localization experiments for imaging of HepG-2 cells with Nile red, a commercially available reagent for probes a and lipid droplets

As shown in FIG. 6, (A) cytographic image of HepG-2 cells treated with only probe a (4.0. mu.M). (B) Imaging of cells treated with nile red only (4.0 μ M). (C) HepG-2 cells with Probe a (4.0. mu.M) and Nile Red (4.0. mu.M). FIG. 7 Probe and Nie Red channel intensity scatter plot, scale: 25 μ M.

A probe channel: the fluorescence emission wavelength of the probe channel is 405nm, and 450-590nm is collected. The fluorescence emission wavelength of the Nie Red channel is 488nm, and 590-620nm is collected. Scale bar: 25 μm.

5. Probe a (4.0. mu.M) stained mouse liver tissue, Living organ and tumor for time-dependent fluorescence imaging of Probe a (see FIG. 8)

Concentration: 10 mu M; λ ex ═ 430nm, λ em ═ 600 nm.

6. FIG. of a biopsy stained with Probe a (4.0. mu.M)

As shown in fig. 9, tumors were imaged using probe a in a tumor-bearing mouse xenograft model prepared by subcutaneously inoculating HepG2 cells. Mice were injected with a (50 μ M, 50mL) in the tumor area and normal area, respectively, and then imaged every 5 minutes.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A fluorescent probe for the specific detection of lipid droplets for distinguishing normal cells and cancer cells is characterized in that the structural formula is as follows:

2. the preparation method of the fluorescent probe that distinguishes normal cells and cancer cells and specifically detects lipid droplets according to claim 1, it is characterized in that the step is as follows: diethyl oxaloacetate and 3-(dimethylamino) Phenol or 3-(diethylamino)phenol was placed in a round-bottomed flask, stirred at 120 °C for 3 h, after cooling, extracted with dichloromethane and water, and purified by silica gel chromatography to obtain the fluorescence of structure a or b. probe.

3. The preparation method of the fluorescent probe for distinguishing normal cells and cancer cells for specific detection of lipid droplets according to claim 2, characterized in that: the diethyl oxalamide acetate and 3-(dimethylamino) The ratio of the amount of phenol or 3-(diethylamino)phenol is 1:1.

4. The application of the fluorescent probe of claim 1 in specifically labeling the morphology of lipid droplets.

5 . The application according to claim 4 , wherein the fluorescent probe can detect lipid droplets in living cells, organs and living bodies, and is used to detect lipid droplet polarity in living systems. 6 .

6 . The application according to claim 4 , wherein the fluorescent probe can image lipid droplets in cancer cells and tumor tissues without background noise, and distinguish normal cells and cancer cells through changes in the number and polarity of image LDs. 7 . cells, normal tissue and tumor tissue.

7. The application according to any one of claims 4-6, wherein the application method is:

(1) Prepare a pH 7.4 PBS buffer solution; weigh the fluorescent probe, dissolve it in DMSO, and accurately prepare a 2mM probe storage solution;

(2) After adding 2 mL of PBS buffer solution to the cuvette, add 4 μL of probe storage solution with a concentration of 2 mM and react with different polar solutions, and excite at 420 nm;

(3) After adding 2 mL of PBS buffer solution to the cuvette, add 4 μL of probe storage solution with a concentration of 2 mM and different ratios of 1,4-dioxane solution, and excite at 420 nm;

(4) Add 2 mL of buffer solutions of different pH to the cuvette, and add 4 μL of the probe storage solution;

(5) Fluorescence imaging of the incubated fluorescent probes in living cells by confocal microscope.

8 . The application according to claim 7 , wherein the living cells in the step (5) are HePG-2 cells, 7702 cells, A549 cells, MCF-7 cells or 3T3 cells. 9 .