CN111253935B - Two-photon fluorescent probe for detecting polarity and viscosity through two channels and preparation method and application thereof - Google Patents
Two-photon fluorescent probe for detecting polarity and viscosity through two channels and preparation method and application thereof Download PDFInfo
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Abstract
The invention disclosesThe two-photon fluorescent probe for detecting polarity and viscosity in two channels, the preparation method and the application thereof can monitor the real-time change of the polarity and the viscosity in cell mitochondria in two different channels at the same time, and the structural formula is as follows:
the two-photon fluorescent probe molecule can utilize two different fluorescent wave bands to simultaneously carry out fluorescent response on polarity (short wave, 410 nm) and viscosity (long wave, 580 nm). In addition, cytotoxicity tests show that the probe has little toxic or side effect on cells, and two-photon confocal fluorescence microscopic imaging experiments show that the probe has good permeability on HeLa cells, can effectively position mitochondria in the cells (the positioning coefficient is 0.97), and is suitable for two-channel two-photon fluorescence imaging and detection of polarity and viscosity in the mitochondria of the cells.
Description
Technical Field
The invention relates to a two-photon fluorescent probe for detecting polarity and viscosity in a two-channel manner, a preparation method and application thereof, which are used for realizing two-channel two-photon imaging and simultaneously detecting polarity and viscosity change in cell mitochondria. Has the advantages of excellent two-photon absorption performance, high membrane permeability, low cytotoxicity, high photostability and biocompatibility.
Background
Cellular and subcellular organelle viscosity is an important microenvironment parameter that exerts biological functions by affecting the interaction and transmission of biomolecules and chemical signals within living cells. Abnormal changes in cell viscosity are closely related to diseases such as diabetes, infarction, hypertension and the like. The polarity of the cell is a feedback of a complex series of mechanisms that establish and maintain the function of a particular domain. Many cellular processes involving spatial arrangement and protein composition, such as differentiation, local membrane growth, immune response activation, directed cell migration, and vector transport of molecules across cell layers, may lead to changes and progression of polarity. Therefore, abnormal changes in polarity are closely related to disorders and diseases.
Mitochondria, which are important organelles in cells as an energy source of the cells, play a key role in cellular metabolism, including supplying metabolic energy through oxidative phosphorylation, and generating and regulating Ca through Reactive Oxygen Species (ROS) 2+ Homeostasis, a cell signaling platform that triggers apoptosis. Notably, the unique function of mitochondria is related to maintaining the homeostasis of its parameters and microenvironment (e.g., pH, viscosity, polarity, temperature, etc.).
The small-molecule fluorescent probe is widely applied to fluorescent imaging due to the advantages of high sensitivity, real-time detection, rapid nondestructive analysis and the like. Measuring subcellular activity of living cells with fluorescent probes is a powerful means to gather information about specific subcellular microenvironments. At present, a plurality of fluorescent probes for detecting the polarity or the viscosity of mitochondria singly exist, but a two-photon fluorescent probe capable of detecting the polarity and the viscosity of mitochondria simultaneously is not reported.
Apoptosis is the most common mode of programmed cell death and is critical for regulating cell balance and maintaining tissue homeostasis. Defects in apoptosis are a major cause of many pathological processes leading to autoimmune diseases and cancer. Thus, one effective therapeutic strategy for the treatment of these diseases, particularly cancer, is to selectively induce cell death by modulating apoptotic pathways. Detection and monitoring of apoptosis is of great interest not only in many cell biology and clinical studies, but also in the assessment of therapy for apoptosis-targeted therapies. From the literature it has been reported that apoptosis causes changes in the intracellular mitochondrial microenvironment such as polarity, viscosity and pH. However, most fluorescent probes can only detect the change of one of the microenvironment parameters, which prevents the dynamic connection between the microenvironments from being understood in a deeper way. Therefore, it is very urgent to develop a two-photon fluorescent probe with excellent performance for dual-channel polarity and viscosity detection, and to understand mitochondrial-related cellular events by monitoring mitochondrial viscosity and polarity changes.
Disclosure of Invention
The invention aims to provide a two-photon fluorescence probe for detecting polarity and viscosity in a two-channel manner, a preparation method and application thereof, and aims to solve the technical problem that a proper fluorescence probe structure is obtained through molecular design, and the two-photon fluorescence probe has the advantages of excellent two-photon absorption performance, low cytotoxicity, good membrane permeability and biocompatibility, high selectivity and light stability and the like, so that the real-time change of polarity and viscosity in the apoptosis process and two-color imaging can be simultaneously detected through two-channel two-photon fluorescence imaging.
The two-photon fluorescent probe is abbreviated as Mito-PV, uses carbazole as a matrix, and has the following structural formula:
the preparation method of the two-photon fluorescent probe comprises the following steps:
step 1: 1-iodohexane (1.56g, 7.38mmol) was added to 2-methylbenzothiazole (1.0 g, 6.71mmol) with stirring at 60 ℃ under nitrogen atmosphere, and cooled to room temperature to give a crude product; the crude product was purified by column chromatography (dichloromethane: methanol =4:1 as eluent) to yield 1.50g (62%) of intermediate 1 — 3-hexyl-2-methylbenzothiazole iodide salt;
step 2: the compound 9-ethyl-6-iodo-9H-carbazole-3-carbaldehyde (1.0g, 2.86mmol), 4-fluoroacetylene (0.41 g, 3.44 mmol), triphenylphosphine palladium dichloride (9.65mg, 0.014mmol), cuprous iodide (5.24mg, 0.027mmol) and triethylamine (2 ml) were reacted under stirring at 30 ℃ under anhydrous and oxygen-free conditions for 12 hours; cooling the mixture to room temperature, precipitating, filtering and concentrating to obtain a crude product; the crude material was purified by column chromatography (petroleum/dichloromethane =4 as eluent) to give intermediate 3- (4-fluorophenylethynyl) -6-carboxaldehyde-N-ethylcarbazole, 0.80g, 82% yield.
And step 3: intermediate 1 (0.32g, 0.9mmol) and intermediate 3 (0.3g, 0.9mmol) are mixed in 50mL of absolute ethanol, the mixture is refluxed for 12 hours under the protection of nitrogen, then cooled, and the solvent is removed through rotary evaporation; the residue obtained was washed with 30mL of saturated brine and then extracted by dichloromethane (3 × 50 mL) and the solvent was evaporated under vacuum; the residue was purified by column chromatography (dichloromethane: methanol =50 as eluent) to give 0.36g (59%) of Mito-PV.
The synthetic route of the two-photon fluorescent probe Mito-PV of the invention is as follows:
the two-photon fluorescent probe is used as a detection reagent when the polarity and the viscosity in mitochondria in cells are detected by double channels. The detection method comprises the following steps:
the two-photon fluorescent probe is dissolved in DMSO to prepare 2mM mother liquor, and 15 mu L of the mother liquor is taken to be placed in 3mL of solvents with different polarities respectively to obtain ultraviolet spectrograms of the probe Mito-PV in the different solvents. Under the excitation of 360nm wavelength, the fluorescence intensity at 410nm gradually decreases along with the increase of the polarity of the test system. Meanwhile, the probe Mito-PV is found to have a stronger fluorescence emission peak only at 580nm in a glycerol solvent with higher viscosity. This suggests that Mito-PV can be used to measure the polarity and viscosity of common solutions. To further verify the dual response characteristics of the probe Mito-PV to polarity and viscosity, the absorption and fluorescence spectra of Mito-PV were first measured in a mixed solvent with different ratios of water and 1, 4-dioxane. When the polarity of the solvent was increased from 10% water (polarity parameter Δ f ≈ 0.229) to 70% water (Δ f ≈ 0.304), only a slight change in the absorption maximum was seen, consistent with the results of measuring different solvent polarities. In contrast, when the polarity (Δ f) of the solution was decreased from 0.304 (70% water) to 0.229 (10% water), the fluorescence intensity of Mito-PV at 410nm increased by a factor of 11. And, the fluorescence intensity of Mito-PV at 410nm has a good linear relationship with Δ f, confirming that Mito-PV has a good fluorescence response to polarity. The fluorescence emission spectra of Mito-PV were then measured in mixed solvents with varying proportions of methanol and glycerol. As the glycerol content of the mixed system was gradually increased (viscosity increased from 1.0 to 300 cp), the fluorescence intensity of Mito-PV at 580nm was increased by a factor of 15, and log I 580nm And log eta has a good linear relation. This indicates that Mito-PV is a solventAlso has good fluorescence response. In addition, the probe Mito-PV is used for testing the change of polarity and viscosity of the etoposide (etoposide) in the process of inducing HeLa cells to die, and with the die of the cells, the fluorescence of a short-wave blue channel is enhanced, which shows that the polarity is reduced, and the fluorescence of a long-wave red channel is enhanced, which shows that the viscosity is increased.
The two-photon fluorescent probe can simultaneously respond to the change of polarity and viscosity by bicolor fluorescence. The cytotoxicity experiment also shows that the probe has lower cytotoxicity, and the two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good permeability to HeLa cells, can effectively position mitochondria in the cells (the positioning coefficients are 0.97 respectively), is suitable for two-photon fluorescence imaging of polarity and viscosity in the mitochondria of the cells, and can detect real-time change and two-color imaging of the polarity and the viscosity in the process of inducing apoptosis by etoposide in a dual-channel manner.
Drawings
FIG. 1 is a (left) UV absorption spectrum of a 10. Mu.M probe in organic solvents of different polarity; (right) fluorescence emission spectra;
FIG. 2 is a graph showing the (left) UV absorption spectra of a 10. Mu.M probe in different volume ratios of water to 1, 4-dioxane mixed solvent; (right) fluorescence emission spectrum, inset: intensity of fluorescence I 410nm And Δ f;
FIG. 3 is a (left) fluorescence emission spectrum of 10. Mu.M probe in different volume ratios of methanol/glycerol mixed solvent; (Right) fluorescence intensity I 580nm And Δ f;
FIG. 4 is a cross-sectional view of effective two-photon absorption of 0.1mM probe in a mixed solvent (left) water/1, 4-dioxane mixed solvent at different volume ratios; (right) mixed solvent of methanol/glycerol with different volume ratios;
FIG. 5 is a graph showing the survival rate of HeLa cells by the action of probe molecules at different concentrations (0. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M).
FIG. 6 is a confocal fluorescence image of location validation of mitochondrial localization of HeLa cells co-stained with 10. Mu.M probe and 1. Mu.M Mitotracker Deep red (MTDR).
FIG. 7 is confocal fluorescence imaging of 10 μ M probe at 50 μ M etoposide-induced HeLa apoptosis.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: synthesis of fluorescent probe molecule Mito-PV
3-hexyl-2-methylbenzothiazole iodide salt (0.32g, 0.9mmol) and 3- (4-fluorophenylethynyl) -6-carboxaldehyde-N-ethylcarbazole (0.3g, 0.9mmol) were mixed in 50mL of anhydrous ethanol; refluxing the mixture under nitrogen for 12 hours, cooling, and removing the solvent by rotary evaporation; the obtained residue was washed with 30mL saturated brine, then extracted by dichloromethane (3 × 50 mL) and the solvent was evaporated under vacuum, and the residue was purified by column chromatography (dichloromethane: methanol =50 as eluent) to give 0.36g (59%) of Mito-PV.
1 H NMR(400MHz,DMSO-d 6 ,ppm):δ9.00(s,1H),8.43(dd,J＝21.2,13.9Hz,3H),8.27(t, J＝9.0Hz,2H),8.03(d,J＝15.6Hz,1H),7.87(dd,J＝8.1,4.9Hz,2H),7.81-7.72(m,3H),7.66 (dd,J＝8.1,5.7Hz,2H),7.31(t,J＝8.7Hz,2H),4.97(t,J＝7.0Hz,2H),4.56(m,J＝6.8Hz,2H), 1.88(dd,J＝15.0,7.7Hz,2H),1.46(dd,J＝14.9,7.2Hz,2H),1.40-1.24(m,7H),0.84(t,J＝7.0 Hz,3H). 13 C NMR(100MHz,DMSO,ppm):δ171.75,160.61,150.62,142.64,141.22,140.07, 133.49,133.41,129.95,129.34,128.79,128.12,127.78,125.69,124.29,123.98,123.81,122.48, 122.41,119.20,116.55,116.14,115.92,113.58,110.64,110.46,110.08,90.04,87.01,48.58,37.67, 30.76,28.65,25.42,21.93,13.85,13.79.
Example 2: spectroscopic testing of fluorescent probe molecules
The two-photon fluorescent probe of the invention is dissolved in DMSO to prepare 2mM mother liquor, 15 mu L of the mother liquor is respectively taken to be put into 3mL of solvents with different polarities, and ultraviolet absorption spectrograms of the probe Mito-PV in different solvents are obtained (the left picture of figure 1). Under the excitation of 360nm wavelength, the fluorescence intensity at 410nm gradually decreases along with the increase of the polarity of the test system. The probe Mito-PV has a strong fluorescence peak only at 580nm in the glycerol solvent with higher viscosity. This indicates that Mito-PV can be used to detect frequentlySee polarity and viscosity of the solution (figure 1, right panel). To further demonstrate the dual response characteristics of polarity and viscosity of the probe Mito-PV, the absorption and fluorescence spectra of Mito-PV were measured at 25 ℃ in mixed solvents with different proportions of water and 1, 4-dioxane (FIG. 2 left panel, FIG. 2 right panel). When the polarity (Δ f) of the solution was decreased from 0.304 (70% water) to 0.229 (10% water), the fluorescence intensity of Mito-PV at 410nm increased by a factor of 11. Also, the fluorescence intensity of Mito-PV at 410nm has a good linear relationship with Δ f (FIG. 2, right panel inset). This indicates that Mito-PV responds well to changes in solvent polarity. The fluorescence spectra of Mito-PV were then measured in mixed solvents with varying proportions of methanol and glycerol (FIG. 2). As the glycerol content in the mixed system was gradually increased (viscosity increased from 1.0 to 300 cp), the fluorescence intensity of Mito-PV at 580nm was increased by a factor of 15 (left panel of FIG. 2), and log I 580nm Has a good linear relationship with log η (figure 2 right). This indicates that Mito-PV has a good fluorescent response to the viscosity of the solvent. The above results indicate that Mito-PV can utilize two different fluorescence bands for detecting the polarity and viscosity of common solutions.
Example 3: two-photon performance testing of fluorescent probe molecules
The effective two-photon absorption cross-sections of Mito-PV in different water and 1, 4-dioxane polar mixed solvents (10%, 40% and 70% water content, respectively) were first tested. The effective two-photon absorption cross-section appeared to be maximum at 720nm and gradually decreased from 88GM to 29GM with increasing water content (left panel of FIG. 3). Mito-PF shows maximum effective two-photon absorption cross section at 840nm in different mixed solvents of methanol and glycerol (glycerol content 99%,80% and 60%, respectively) and gradually decreases from 96GM to 48GM as the glycerol content decreases (right diagram of FIG. 3). Mito-PV has the ability to be used for two-photon bioimaging of intracellular polarity and viscosity as demonstrated above.
Example 4: cytotoxicity test
We performed cytotoxicity experiments using the MTT (5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) method. The Mito-PV is added into live HeLa cells at various concentrations (0 muM, 10.0 muM, 20.0 muM, 30.0 muM), and the results of the test after 24 hours are shown in figure 4, and when the concentration of the probe reaches 30 muM, the survival rate of the cells still has about 90 percent, which indicates that the fluorescent probe molecule has no obvious toxic effect on the cells and can be applied to biology.
Example 5: cell localization assay
To investigate the mitochondrial localization properties of Mito-V, we used here the commercial dye Mitotracker Deep (MTDR) to co-localize with Mito-PV in HeLa cells. The results show the yellow channel (. Lamda.) of Mito-PV em ＝ 500-600nm，λ ex =720 nm) and MTDR (λ) em ＝655-755nm,λ ex =635 nm), and the Pearson co-localization coefficient of Mito-PV and MTDR was calculated to be 0.97 (fig. 5). These results indicate that Mito-PF can be well localized in the mitochondria of living cells.
Example 6: confocal fluorescence imaging of apoptosis
Etoposide (etoposide) is a recognized apoptosis agent that can cause apoptosis. From the literature, it is reported that apoptosis causes changes in the intracellular mitochondrial microenvironment, such as polarity, viscosity and pH. Therefore, we attempted to use Mito-PV to monitor real-time changes in mitochondrial polarity and viscosity during etoposide-induced apoptosis. 10 μ M Mito-PV was added to the cells and incubated for 0.5h. Etoposide (50 μ M) was then added to the cells and imaging was performed every 15 minutes. Can be found in Blue Channel (lambda) by imaging em =420-460 nm), indicating a gradual decrease in polarity. And Red Channel (λ) em ＝500-600nm，λ ex =720 nm), indicating a gradual increase in viscosity. From the analysis of the above data, we can clearly find that as the time of adding etoposide (etoposide) increases, i.e. apoptosis gets deeper, the polarity of mitochondria in cells gradually decreases and the viscosity gradually increases. This suggests that etoposide-induced apoptosis can cause a decrease in mitochondrial polarity and an increase in viscosity within the cell.
Claims (3)
1. A two-photon fluorescent probe for detecting polarity and viscosity by two channels is abbreviated as Mito-PV, and is characterized in that the structural formula is as follows:
2. a method for preparing the two-photon fluorescent probe according to claim 1, which comprises the steps of:
step 1: adding 1-iodohexane into 2-methylbenzothiazole under stirring at 60 ℃ in a nitrogen atmosphere, and cooling to room temperature to obtain a crude product; purifying the crude product by column chromatography to obtain intermediate 1-3-hexyl-2-methylbenzothiazole iodide salt;
step 2: stirring and reacting a compound 9-ethyl-6-iodine-9H-carbazole-3-formaldehyde, 4-fluoroacetylene, triphenylphosphine palladium dichloride, cuprous iodide and triethylamine for 12 hours at the temperature of 30 ℃ under anhydrous and oxygen-free conditions; cooling the mixture to room temperature, precipitating, filtering and concentrating to obtain a crude product; purifying the crude material by column chromatography to obtain an intermediate 3- (4-fluorophenylethynyl) -6-formyl-N-ethylcarbazole;
and step 3: mixing the intermediate 1 and the intermediate 3 in absolute ethyl alcohol, carrying out reflux reaction for 12 hours under the protection of nitrogen, then cooling, and removing the solvent through rotary evaporation; the residue obtained was washed with saturated brine, then extracted with dichloromethane and the solvent was evaporated under vacuum; purifying the residue by column chromatography to obtain a target product Mito-PV;
in step 1, the eluent for column chromatography purification is dichloromethane: methanol =4:1,v/v;
in step 3, the eluent for column chromatography purification is dichloromethane: methanol =50:1,v/v.
3. Use of the two-photon fluorescence probe according to claim 1, wherein: the two-photon fluorescent probe is used as a detection reagent when detecting the polarity and viscosity in mitochondria in cells; the two-photon fluorescent probe can simultaneously respond to the change of polarity and viscosity by bicolor fluorescence.
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