CN115232158B

CN115232158B - Fluorescent probe for detecting polarity of targeted lipid droplets and preparation method and application thereof

Info

Publication number: CN115232158B
Application number: CN202210527831.9A
Authority: CN
Inventors: 林伟英; 唐永和; 宋思睿
Original assignee: Guangxi University
Current assignee: Guangxi University
Filing date: 2022-05-16
Publication date: 2024-07-09
Anticipated expiration: 2042-05-16

Abstract

The invention provides a fluorescent probe for detecting polarity of a targeted lipid drop, which is called SG-LDs for short, and has a chemical structural formula of; The fluorescent probe has the advantages of simple synthesis process, capability of targeting lipid drops, sensitivity to polarity, low cytotoxicity, capability of imaging in cells and tissues, high light stability and wide application range, and is a novel near infrared probe.

Description

Fluorescent probe for detecting polarity of targeted lipid droplets and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting polarity of a targeted lipid droplet and application thereof.

Background

In biology, the polarity of the cellular microenvironment is one of the key factors affecting various cellular processes. Some cellular processes, which are related to spatial arrangement and protein composition, have an inseparable relationship to polarity, such as cell differentiation, membrane growth of individual subcellular organelles, and transmembrane transport. Importantly, polarity changes in real time as the state of the cell changes, and abnormalities in polarity often lead to the onset of diseases such as: diabetes, depression, and the like. Therefore, detection of changes in polarity of biological microenvironments is of critical importance in studying the occurrence and diagnosis of these diseases. Although the methods for detecting the polarity of biological microenvironments have been diverse and rapidly developed in recent years, precise and comprehensive techniques remain lacking.

Lipid Droplets (LDs) are lipid subcellular organelles essential for cell metabolism and energy storage, which are encapsulated in phospholipid layers, ubiquitous in various cell types, and involved in various physiological processes. Lipid droplets have long been ignored as a lipid organelle, and in fact are closely related to many physiological activities. The occurrence of some metabolic diseases is not separated from the change of the number and polarity of lipid droplets, such as fatty liver, liver injury and hepatitis. In the early stage of liver diseases, the metabolism of lipid is obviously up-regulated, LDs can accumulate in cells except fat cells, so that the polarity of lipid droplets of pathological cells is obviously lower than that of normal cells, but the number of lipid droplets is greatly increased. Therefore, detecting whether the number and polarity of lipid droplets are abnormal can distinguish normal cells from diseased cells, and finding a method capable of targeting and monitoring lipid droplets in real time is particularly important for early prediction of cytopathy and diseases.

The fluorescent probe has high sensitivity, can realize real-time monitoring while targeting and positioning, attracts high attention of researchers, and is widely applied to detection of small molecules or microenvironments in living systems. Whereas near infrared fluorescent probes can penetrate living tissue and can inhibit photodamage to cells and tissues, some diseases can be visualized for in vivo detection. Currently Bodipy493/503 green and Nile Red are the most commercially used LDs fluorescent dyes. However, the lack of specificity of Bodipy493/503 green and Nile Red limits the use of these two probes at the cellular level and does not recognize tissue microstructure.

Disclosure of Invention

Aiming at the problem of poor specificity of lipid drop probes in the prior art, the invention provides a fluorescent probe for detecting polarity by targeting lipid drops, which is sensitive to polarity and low in toxicity, can be imaged in cells, tissues and organisms, and has wide application prospect.

Another object of the present invention is to provide an application of the above fluorescent probe in detecting the polarity of a solution, a cell or a tissue.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

A fluorescent probe for detecting polarity of a targeted lipid drop is called SG-LDs for short, and the chemical structural formula of the fluorescent probe is shown as formula (I):

formula (I).

The preparation method of the fluorescent probe comprises the following steps:

(1) 4- (diethylamino) -salicylaldehyde and 4-hydroxy-6-methyl-pyrone react in ethanol under the catalysis of piperidine in a protective atmosphere, the solid-liquid separation of the reaction mixture is carried out, and the solid product is SG-1:

；

(2) SG-1 and boron trifluoride-diethyl etherate react in methylene dichloride in protective atmosphere, the solid-liquid separation of reaction mixture, solid purification get SG-2:

；

(3) SG-2 and 4-dimethylamino benzaldehyde react in toluene under the catalysis of piperidine, and after the reaction is finished, a blue compound is obtained through separation and purification, namely, the fluorescent probe is obtained:

。

In the step (1), the molar ratio of the 4- (diethylamino) -salicylaldehyde to the 4-hydroxy-6-methyl-pyrone is 4:3.

In step (1), the reaction temperature was 80 ℃.

In the step (2), the molar ratio of SG-1 to boron trifluoride-diethyl etherate is 1:1.

In the step (2), the reaction temperature is 0-4 ℃.

In step (2), the purification step is to recrystallize the solid from chloroform.

In the step (3), the molar ratio of the SG-2 to the 4-dimethylaminobenzaldehyde is 1.5:1

In the step (3), the reaction temperature is 20-30 ℃.

In the step (3), the reaction liquid is spin-dried, dissolved by methylene dichloride, washed by water, the organic phase is taken after layering, concentrated and chromatographed by a silica gel column, methylene dichloride and petroleum ether with the volume ratio of 2:1 are taken as eluent, and the eluent is distilled under reduced pressure.

Use of a fluorescent probe as described above for detecting the polarity of a solution, lipid droplets or polarity in a cell, tissue or organism.

The mechanism of the invention is as follows:

In the probe molecule, dimethylaminophenyl is an electron-donating group, and can form a strong intramolecular electron-pushing system in the molecule, so that an excited state Intramolecular Charge Transfer (ICT) process is caused, and the probe molecule is fluorescent. In the case of increasing polarity, the excited state energy of the probe is rapidly dissipated in the solvent through dipole-dipole interaction, so that the probe has a longer wavelength and lower fluorescence intensity in a high-polarity environment, and the blue shift phenomenon of the emitted light wavelength of the probe molecule occurs and the fluorescence intensity is enhanced with the decrease of the polarity of the surrounding environment.

The invention has the following advantages:

therefore, a new polarity sensitive fluorescent probe SG-LDs of the near infrared targeting lipid drop is designed according to the targeting positioning capability of the fluorescent probe. The probe is subjected to optical analysis by spectral test, and the polarities of different solvents in a microenvironment can be accurately analyzed, so that the probe is proved to be sensitive to the polarities; cytotoxicity test proves that the probe has weak toxicity and can be used in clinical medicine; cell and tissue imaging (mice normal liver, fatty liver, injured liver and hepatitis mice) tests, the probe can well target lipid droplets and observe the polarity change, thereby distinguishing diseased cells from normal cells.

Compared with other positioning lipid drop probes, the probe is a novel near infrared probe, has strong light stability and wide application range.

Drawings

FIG. 1 is a hydrogen spectrum of a fluorescent probe;

FIG. 2 is a carbon spectrum of a fluorescent probe;

FIG. 3 is a mass spectrum of a fluorescent probe;

FIG. 4 is a cytotoxicity experiment of fluorescent probes in different cells;

FIG. 5 is a reaction of a fluorescent probe to solutions of different polarities;

FIG. 6 is a graph showing selectivity of fluorescent probes for different substances;

FIG. 7 is an image of fluorescent probes in cells of different polarity;

FIG. 8 is an image of lipid droplets in a fluorescent probe and bodipy located in different cells;

FIG. 9 is an imaging application of fluorescent probes in zebra fish;

FIG. 10 is a fluorescent probe for use in mouse liver imaging.

Detailed Description

The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.

Example 1 Synthesis of fluorescent probes

(1) Dissolving 4- (diethylamino) -salicylaldehyde (3.829 g,30.3 mmol g,40 mmol) and 4-hydroxy-6-methyl-pyrone (5 g,40 mmol) in 50mL ethanol under nitrogen protection, stirring and heating to 80deg.C, adding a certain amount of piperidine into the system during heating, refluxing for 4h, and filtering when orange crystal is precipitated in the system to obtain solid product SG-1(2.3g,30%),¹H NMR (500 MHz, CDCl₃-d) δ 8.52 (s, 1H), 7.40 (dd, J = 8.9 Hz, 1H), 6.98 (s, 1H), 6.66 (dd, J₁= 9.0 Hz, J₂ = 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 3.48 (q, J = 7.2 Hz, 4H), 2.22 (s, 3H), 1.25 (t, J = 7.1 Hz, 6H)：

；

(2) Under the protection of nitrogen, 10mL of anhydrous dichloromethane is added dropwise to SG-1 (600 mg,2 mmol) to dissolve the water, 8g of boron trifluoride diethyl etherate is added into the system under the ice bath condition, white solid is separated out along with the reaction, the reaction is carried out for 8 hours, suction filtration is carried out, the crude product is obtained, and then chloroform is used for recrystallization, thus obtaining solid SG-2(599mg,86%),¹H NMR (500 MHz, CDCl₃-d) δ 8.79 (s, 1H), 7.45 (d, J = 10 Hz, 1H), 7.44 (s, 1H), 6.70 (dd, J₁ = 10 Hz, J₂= 2.3 Hz, 1H), 6.47 (d, J = 2.3 Hz, 1H), 3.51 (q, J = 7.2 Hz, 4H), 2.39 (s, 3H), 1.28 (t, J = 7.2 Hz, 6H)：

；

(3) SG-2 (300 mg,0.62 mmol) was dissolved in 10mL toluene, a catalytic amount of piperidine was added to the system, after stirring at room temperature for a period of time, 4-dimethylaminobenzaldehyde (300 mg,2 mmol) was added, the reaction system became purplish red over time, the mixture was dried after 24h of reaction, dissolved in methylene chloride, washed with water, the organic phase was concentrated after separation, and then chromatographed on a silica gel column with 2:1 volume ratio of methylene chloride and petroleum ether as eluent, the eluent was distilled under reduced pressure to give a blue compound, i.e., a fluorescent probe (100 mg, yield 33%):

。

Its ¹ H NMR spectrum is shown in figure 1：¹H NMR (500 MHz, DMSO-d₆) δ 8.77 (s, 1H), 7.91 (d, J = 15.2 Hz, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.5 (d, J = 9.5 Hz, 2H), 7.39 (s, 1H), 6.98 (d, J = 15.2 Hz, 1H), 6.88 (dd, J₁= 9.2 Hz, J₂= 2.4 Hz, 1H), 6.78 (d, J = 9.05 Hz, 2H), 6.66 (d, J = 2.1 Hz, 1H), 3.56 (q, J = 7.0 Hz, 4H), 3.08 (s, 6H), 1.18 (t, J = 7.1 Hz, 6H);

Its ¹³ C NMR spectrum is shown in figure 2：¹³ C NMR (125 MHz, DMSO- d₆) δ 179.17, 172.68, 159.11, 158.69, 154.64, 153.63, 148.16, 147.74, 133.66, 132.95, 122.24, 115.34, 112.52, 111.55, 109.41, 108.29, 100.13, 96.48, 45.27, 12.93;

The mass spectrum is shown in figure 3：HR-MS (ESI) calcd. for C₂₆H₂₈BF₂N₂O₄ ⁺ [M+H]⁺ m/z 481.2219, found 481.2214;

Fluorescence quantum yield of this probe in different solvents table 1 shows:

TABLE 1 Quantum yield of fluorescent probes

Epsilon-molar absorption coefficient; lambda _a -maximum absorption wavelength; lambda _b -maximum emission wavelength; phi _Fl -fluorescence quantum yield.

Example 2 response of fluorescent probes to solutions of different polarity

3ML of a fluorescent probe stock solution having a concentration of 1mM was prepared for use. Preparing probe concentration of 10 mu M, respectively interacting with PBS buffer solution (10 mmol, pH=7.4) and mixed solution (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100%) of 1, 4-dioxane, setting excitation wavelength of 560nm, detecting fluorescence with detection wavelength of 600-850nm, calculating fluorescence intensity in each system, and establishing a curve related to the proportion of fluorescence intensity to polarity, wherein the result is shown in FIG. 4: as the polarity is reduced, the fluorescence intensity of the reaction system is gradually increased.

Example 3 selectivity of fluorescent probes for different active Agents

The probe concentration was 10. Mu.M, and different positive ions, negative ions and amino acids (：Cys、GSH、Hcy、F^-、SO₄ ^2-、Cu²⁺、ClO^-、Ca²⁺、H₂O₂、I^-、Mg²⁺、Mn²⁺、Na₂S、Zn²⁺、 methionine, t-butyl hydroperoxide, arginine, lysine, valine, glycerol, DNA1 (TGAGGTAGTAGGTTGTATAGTT), DNA2 (AACTATACAACCTACTACCTCAGAGTCAGTCATATCTAT) and bovine serum albumin, respectively) were added to give a final concentration of 10mM. Fluorescence detection was performed with excitation wavelength of 560nm and detection wavelength of 580nm to 750nm, and the results are shown in FIG. 5: under the condition of adding different active substances, the fluorescence signal intensity of the SG-LDs is almost unchanged. This indicates that SG-LDs respond negligible to these active species relative to the sensitivity of the probe pair to polarity.

Example 4 toxicity of fluorescent probes to cells

Cytotoxicity experiments were performed on four cells (Hela, HL7702,4T1 and 3T 3): culture media containing different concentrations of probes (1, 2, 5, 10, 20, 30, 40. Mu.M) were added to 96-well plates containing cells for 24 hours of incubation, followed by examination of the viability of the cells. As can be seen from fig. 6, the survival rate of cells is as high as 90% at all concentrations, and regrowth occurs even at lower concentrations.

Application example 1 imaging of fluorescent probes in living cells of different polarities

Four cells (4T 1,3T3, hela, HL7702) were inoculated into 35mm glass-bottomed cell culture dishes, cultured in a carbon dioxide incubator (temperature 37 ℃,5% CO ₂), incubated for 30min with 10. Mu.L SG-LDs after the cells had adhered to the wall, washed 3 times with PBS buffer after the end of the culture, and fluorescent photographs of the four cells were taken with a fluorescence microscope, respectively, with lambda _ex =580 nm, and the collection band at 610nm-750nm, as shown in FIG. 7: the first column: a bright field; the second column: a red channel; third column: a superimposed field; a first row: HL7702; a second row: hela; third row: 3T3; fourth row: 4T1. The fluorescent probe was found to emit intense fluorescence, indicating that the fluorescent probe was able to distinguish normal cells from cancer cells.

Application example 2 localization of fluorescent probes to lipid droplets in different cells

Cancer cells at a density of 3×10 ⁵/mL were inoculated into 35mm glass-bottomed cell culture dishes and two groups of cells were cultured in a carbon dioxide incubator (37 ℃,5% co ₂), one of which was incubated 24 h with 10 mmol oleic acid. After cell attachment, experimental group culture was performed by adding 200nM of lipocalin dye bodipy to cells, incubating for 20min, washing the cells 3 times with PBS buffer, then adding 10. Mu.M probe SG-LDs to a cell culture dish, incubating for 30min in a cell incubator, washing the cells 3 times with PBS buffer, and taking fluorescence photographs of the cells with a fluorescence microscope, respectively, the results are shown in FIG. 8, wherein: a) Co-localization imaging of HeLa cells; b) Co-localized imaging of 3T3 cells; c) Co-localized imaging of 4T1 cells; in each group, 1 is the red channel of SG-LDs; 2 is the green channel of bodipy; 3 is a bright field channel; 4 is the superposition of the three figures; 5 is the intensity distribution of SG-LDs in the red channel and bodipy in the green channel. Green channel: lambda _ex =580 nm, collection band is 610nm-750nm, red channel: lambda _ex =488 nm, the collection band is 500nm-550nm. The localization coefficients of HeLa cells, 3T3 cells and 4T1 cells are 94.69%, 94.02% and 90.64%, respectively, and the cell localization Coefficient Pearson's Coefficient in three cells is more than 90%, which indicates that the probe can well localize lipid droplets.

Application example 3 imaging application of fluorescent probe in zebra fish

Putting a group of zebra fish into a culture solution containing 1mM oleic acid in advance for incubation for 24 hours, and increasing the number of LDs in the zebra fish; the other group was fed normally. SG-LDs (10. Mu.M) were then incubated with zebra fish for 20min, and fluorescence photographs of the zebra fish cultured under these two groups of conditions were taken with a fluorescence microscope, respectively, and the results are shown in FIG. 9: the first column: a bright field; the second column: a red channel; third column: a superimposed field; a first row: zebra fish with probes only; a second row: zebra fish with probe and oleic acid. Zebra fish added with oleic acid was found to emit a distinct fluorescent signal.

Application example 4 imaging application of fluorescent probe in mouse liver cells

The same-age Kunming mice (female mice) are divided into two groups, one group is fed with high-fat feed for three days, a fatty liver model is built, and the other group is fed with common feed for three days, so that a blank control experiment is realized. The livers in mice were taken out and immersed in 10. Mu.M SG-LDs solution for 6 hours, and then fluorescence imaging was performed at an excitation wavelength of 600nm and an emission wavelength of 660nm, as shown in FIG. 10: the fluorescence intensity of the disease liver model (2-3) is obviously much stronger than that of the common liver model (1). The experimental result shows that SG-LDs has application potential in fatty liver diagnosis.

Claims

1. Use of a fluorescent probe according to formula (I) for the preparation of a reagent for detecting the polarity of a solution, lipid droplets or polarity in cells, tissues or organisms

Formula (I).