CN114702507B - Fluorescent probe for detecting lipid droplets and endoplasmic reticulum - Google Patents
Fluorescent probe for detecting lipid droplets and endoplasmic reticulum Download PDFInfo
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- CN114702507B CN114702507B CN202210559646.8A CN202210559646A CN114702507B CN 114702507 B CN114702507 B CN 114702507B CN 202210559646 A CN202210559646 A CN 202210559646A CN 114702507 B CN114702507 B CN 114702507B
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention provides a fluorescent probe capable of detecting intracellular lipid droplets and endoplasmic reticulum, which is formed by jointly constructing a rhodamine dye part and a coumarin dye part, is based on an intramolecular reversible ring-opening/closing reaction mechanism, and can distinguish Lipid Droplets (LDs) and Endoplasmic Reticulum (ER) in double emission channels. The spectrum difference of the fluorescent probe is up to 320 nm, organelles can be distinguished between two emission channels without mutual interference, and an important molecular tool is provided for promoting the research of the interaction of the organelles.
Description
Technical Field
The invention belongs to the technical field of material science, and particularly relates to a fluorescent probe for detecting lipid droplets and endoplasmic reticulum.
Background
Organelles within eukaryotic cells are often separated by membrane systems to perform specific biological functions. These organelles are not independent, and they cooperate and communicate with each other closely to achieve many important biological processes such as rapid material exchange, information transfer, etc. Therefore, communication and interaction between organelles is an important topic for research. Lipid Droplets (LDs) are specialized phospholipid monolayer-encapsulated organelles derived from the Endoplasmic Reticulum (ER) and are responsible for storing neutral lipids such as triglycerides and cholesterol esters. Lipid droplets play an important role in maintaining lipid homeostasis in cells, and dysregulation of lipid droplets can lead to various diseases such as obesity, fatty liver, and the like.
The endoplasmic reticulum is an important organelle widely existing in eukaryotic cells and is the site for synthetases, lipids and transmembrane proteins, and the interaction of lipid droplets with the endoplasmic reticulum can promote the efficient transport of lipid droplet-associated proteins, neutral lipids and phospholipids from the endoplasmic reticulum to the lipid droplets, and at the same time, the lipid droplets reduce lipid toxicity by collecting free lipids generated during endoplasmic reticulum stress and autophagy. Defects in the interaction of lipid droplets and the endoplasmic reticulum can lead to serious diseases including lipodystrophy and nervous system diseases. Therefore, visualizing the interaction between lipid droplets and the endoplasmic reticulum is crucial for elucidating and revealing the underlying mechanisms of biology and pathology.
The interaction between the two organelles can be visualized by various methods, including transmission electron microscopy, scanning electron microscopy, immunofluorescence microscopy, and the like. These methods provide important tools for revealing interactions between organelles. However, these methods require fixation of the biological sample, which inevitably causes damage to the sample. Compared with these methods, the fluorescence imaging technology is widely used due to its advantages of simple operation, small damage to living cells, proper resolution, real-time observation, etc. Fluorescent probes, which are capable of displaying two organelles simultaneously, are promising tools for studying organelle interactions.
To reveal the interaction between the lipid droplet and the endoplasmic reticulum, the two organelles can optionally be labeled with different emission colors with fluorescent probes. However, such fluorescent probes have been developed only rarely, and the reported probes have certain disadvantages. For example, guo et al (Journal of the American Chemical Society 2021, 143 (8), 3169-3179.) provide the first fluorescent probes for two-color fluorescence imaging of lipid droplets and endoplasmic reticulum based on an Excited State Intramolecular Proton Transfer (ESIPT) mechanism, however, the emission spectra of the probes in lipid droplets and endoplasmic reticulum show significant interference. Although the probe plays a crucial role in the study of lipid droplet and endoplasmic reticulum interaction, the sensitivity needs to be improved to improve accuracy. Therefore, it is of great interest to develop fluorescent probes that can identify lipid droplets and endoplasmic reticulum that are not interfered with in different fluorescence channels.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fluorescent probe for detecting lipid droplets and endoplasmic reticulum, wherein the fluorescent probe is CCA, and the molecular structural formula is as follows:
the preparation method of the fluorescent probe comprises the following steps:
s1, refluxing 4-diethylamino keto acid and cyclohexanone in concentrated sulfuric acid for 6 hours, cooling to room temperature, pouring into ice water, adding perchloric acid to generate a solid, and filtering to obtain a compound 1;
s2: and refluxing the compound 1 and the compound 2 in acetic anhydride for 12 hours, cooling to room temperature, filtering to obtain a crude product, and purifying by using a silica gel chromatographic column to obtain the fluorescent probe CCA.
Another object of the invention is to provide an application of a fluorescent probe for detecting lipid droplets and endoplasmic reticulum in preparing a lipid droplet and endoplasmic reticulum probe for simultaneously imaging cells or tissues in two colors.
The working principle of the fluorescent probe is as follows: by utilizing the reversible ring-opening/closing reaction of rhodamine dye, CCA is emitted in a ring-closing green mode in an aprotic solvent and is emitted in a ring-opening near infrared mode under the condition of certain water content. The core of the lipid droplets is highly hydrophobic with negligible water content, while the endoplasmic reticulum membrane contains some amount of water. Thus, CCA labels lipid droplets and endoplasmic reticulum in green and near-infrared fluorescence with spectral differences up to 320 nm.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) The fluorescent probe obtained by the invention can simultaneously distinguish and image lipid drops and endoplasmic reticulum in two colors based on the intramolecular reversible open-loop closed reaction, and is favorable for intuitively researching the interaction between double organelles;
(2) The fluorescent probe of the invention emits green fluorescence in a ring-closed manner in the lipid drop and emits near-infrared fluorescence in a ring-open manner in the endoplasmic reticulum. The spectral difference is as high as 320 nm, so that the two emission channels do not interfere with each other
(3) The fluorescent probe obtained by the invention has the advantages of sensitive detection, quick response, strong light stability and accurate positioning.
Detailed Description
In order to make the purpose and technical solution of the present invention more clear, the present invention is further described with reference to the following examples, but the scope of the present invention is not limited to these examples, and the examples are only used for explaining the present invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true scope of the invention. The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
The chemicals used in the present invention were analytical grade, 2- (4-diethylamino-2-hydroxybenzoyl) benzoic acid and cyclohexanone were purchased from national pharmaceutical chemicals, ltd (shanghai, china). 4- (diethylamino) -2-hydroxybenzaldehyde, diethyl malonate, etc. were purchased from Beijing J & K chemical company. The solvents used in the spectroscopic measurements are of chromatographic grade.
Example 1:
synthesis of Compound 1
6 mL of concentrated sulfuric acid was added to a round-bottom flask, cooled to 0 deg.C, and cyclohexanone (0.59 g, 6 mmol) was added dropwise. 2- (4-diethylamino-2-hydroxybenzoyl) benzoic acid (0.94 g, 3 mmol) was then added with stirring. The reaction was heated to reflux for 6 hours, then cooled to temperature and poured into ice water. Then, about 5mL of perchloric acid was added dropwise. Filtered and used in the next reaction without further purification.
Synthesis of Probe CCA:
in a round-bottom flask, compound 1 (0.48 g, 1 mmol) and compound 2 (0.25 g, 1 mmol) were added, and 4 mL of acetic anhydride was added. The reaction was heated to reflux for 12 hours, cooled to room temperature and the mixture was poured into ice water. The product was extracted three times with dichloromethane and the solvent was concentrated to give the crude product. With CH 2 Cl 2 Purification by silica gel chromatography with MeOH (V: V = 10) as eluent gave the product as a brown powder (0.4 g, 54.8% yield). 1 H NMR (400 MHz, MeOD) δ 8.17 (d,J= 6.3 Hz, 1H), 8.06 (d,J= 15.5 Hz, 2H), 7.74 – 7.64 (m, 2H), 7.51 (d,J= 8.9 Hz, 1H), 7.24 (d,J= 7.8 Hz, 1H), 7.09 (d,J= 21.1 Hz, 3H), 6.79 (dd,J= 9.0, 2.0 Hz, 1H), 6.55 (s, 1H), 5.51 (s, 1H), 3.69 (q,J= 7.0 Hz, 4H), 3.54 (q,J= 7.0 Hz, 4H), 2.97 (s, 2H), 2.45 (d,J= 29.3 Hz, 2H), 1.87 (s, 2H), 1.32 (t,J= 7.0 Hz, 6H), 1.25 (t,J= 7.0 Hz, 5H). 13 C NMR (101 MHz, MeOD) δ 171.44 (s), 162.51 (s), 159.56 (s), 157.91 (s), 156.33 (s), 155.00 (s), 152.14 (s), 143.66 (s), 137.29(s), 135.29 (s), 130.61 (s), 130.21 (d,J= 14.9 Hz), 129.54 (s), 129.34 (s), 128.89 (s), 128.26 (s), 127.27 (s), 121.71 (s), 116.20 (d,J= 11.9 Hz), 114.78 (s), 109.93 (s), 108.79 (s), 96.34 (s), 95.08 (s), 45.37 (s), 44.58(s), 27.29 (s), 25.33 (s), 21.11 (s), 11.49 (d,J= 5.0 Hz) (nuclear magnetic results see FIG. 1, FIG. 2)
Example 2: cellular imaging of fluorescent probe CCA.
(1) Cell culture
HepG2 cells were purchased from Procell Life Science&Technology Co., ltd., in H-DMEM (Dulbecco's modified Eagle's Medium, high Glucose) Medium containing 10% fetal bovine serum, in 5% CO 2 Culturing at 37 ℃ in an incubator. For cell imaging experiments, live HepG2 cells were diluted in 10000 cells/mL of medium. 1 mL of the cell suspension was added to a glass petri dish and cultured for 24 h to allow the cells to adhere.
(2) Cell imaging experiments
Live HepG2 cells were adhered by culturing for more than 24 hours in glass-bottom dishes. The medium was removed, 1 mL of fresh medium was incubated with 10. Mu.M probe CCA for 30 min, and cells were observed by Nikon A1R microscopy. For the green channel, λ ex = 405 nm， λ em 500-550 nm and near infrared channel lambda ex = 647 nm; λ em = 663-738 nm。
As shown in fig. 3, bright fluorescent signals were detected in both the green channel and the near infrared channel. Meanwhile, the green emission is in a point shape, the near infrared fluorescence forms a net shape, which shows that the CCA marks lipid droplets and endoplasmic reticulum in the green and near infrared emission, and the fluorescence signals of the green channel and the near infrared channel never overlap, which shows that the interference between the two channels can be ignored.
The magnified image of fig. 4 shows that the green fluorescent signal is distributed mainly in the cytoplasm and overlaps with the black spot. These black dots may be designated as lipid droplets, which appear as black dots or bright dots in DIC images because they are composed of neutral lipids having a higher refractive index than water.
As shown in figure 5, because the distribution of endoplasmic reticulum and lipid droplets in different tissues is different greatly, the distribution of endoplasmic reticulum and lipid droplets is observed by staining mouse heart, liver, lung and adipose tissues with CCA, and the myocardial green emitter is weak and the near infrared fluorescence is strong, which shows that the myocardium contains almost no lipid droplets. At the same time, the liver showed several small green emission points and strong near-infrared fluorescence, indicating that the number of lipid droplets in the liver of healthy mice is very limited. In lung tissue, intense fluorescent signals were detected in both the green and near-infrared channels, suggesting the presence of a large number of lipid droplets and endoplasmic reticulum in lung tissue. The distribution of lipid droplets and endoplasmic reticulum in white adipose tissue was observed. It can be seen that the distribution of lipid droplets and endoplasmic reticulum in white adipocytes is very regular. The cell has a large lipid droplet in the center and an endoplasmic reticulum surrounding it.
Claims (3)
1. A fluorescent probe for detecting intracellular lipid droplets and endoplasmic reticulum is characterized in that the code of the fluorescent probe is CCA, and the molecular structural formula is as follows:
2. a method for preparing the fluorescent probe of claim 1, wherein the method comprises:
s1: refluxing 4-diethylamino keto acid and cyclohexanone in concentrated sulfuric acid to obtain a compound 1;
s2: and refluxing the compound 1 and the compound 2 in acetic anhydride to obtain the fluorescent probe CCA.
3. Use of the fluorescent probe for detecting lipid droplets and endoplasmic reticulum in cells or tissues according to claim 1 for the preparation of a probe for simultaneous two-color imaging of lipid droplets and endoplasmic reticulum in cells or tissues.
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| CN112341419A (en) * | 2020-11-10 | 2021-02-09 | 山东大学 | Single-molecule fluorescent probe capable of distinguishing imaging lipid drop and endoplasmic reticulum simultaneously and application thereof |
| CN112250652A (en) * | 2020-11-10 | 2021-01-22 | 山东大学 | Single-molecule fluorescent probe capable of simultaneously performing two-color imaging on lipid droplets and endoplasmic reticulum and application thereof |
| CN114292281B (en) * | 2021-12-22 | 2023-10-24 | 东南大学 | Small-molecule fluorescent probe for realizing dynamic imaging of intracellular lipid droplets, and preparation method and application thereof |
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