CN108329301B

CN108329301B - Two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells and preparation method and application thereof

Info

Publication number: CN108329301B
Application number: CN201810256444.XA
Authority: CN
Inventors: 孟祥明; 候立玲; 宁鹏; 程浩然; 冯燕
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2020-10-02
Anticipated expiration: 2038-03-27
Also published as: CN108329301A

Abstract

The invention discloses a two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells, a preparation method and application thereof, wherein the two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells has the following structure:

the fluorescent probe can respond to a fluorescent signal with specificity to pH. The cell co-localization experiment proves that the probe can specifically target cell lysosomes, cytotoxicity test shows that the probe has little toxic or side effect on cells, and two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good permeability on MCF-7 cells, and meanwhile, the pKa of the fluorescent probe is calculated to be 3.88, so that the probe is very suitable for monitoring the pH variation range of the cell lysosomes, and the condition of the cell autophagy process can be monitored in real time by detecting the pH variation of the cell lysosomes.

Description

Two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells and preparation method and application thereof

Technical Field

The invention relates to a two-photon ratio type fluorescent probe, in particular to a two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells and a preparation method and application thereof.

Background

Autophagy is the process by which cells, when subjected to destructive stimulation, phagocytose self-cytoplasmic proteins or organelles and invaginate into vesicles which are then fused with lysosomes to form autophagosomes, degrading the contents they encapsulate. The autophagy of the cells is the basis of the degradation and recycling of cell components, plays a role of 'scavenger' in the cells, and is used as a normal way for natural metabolism of intracellular organelles and other structures, when histiocytes are damaged by various physicochemical factors, autophagy lysosomes are greatly increased, thereby playing a role in protecting the damage of the cells. Traditional autophagy monitoring includes Transmission Electron Microscopy (TEM), Western blotting (Atg8/LC3) and GFP-Atg8/LC3 fluorescence microscopy. However, these monitoring methods have limitations, such as scanning electron microscopy and western blotting, and they cannot monitor the autophagy status in living cells, and the results of these methods are not ideal, so that a method capable of monitoring autophagy vividly and easily is necessary to study the autophagy process of cells.

Lysosomes are organelles of a single-layer membrane sac-like structure in eukaryotic cells, contain a plurality of hydrolytic enzymes, mainly play a role in decomposing substances entering the cells from the outside and digesting local cytoplasm or organelles of the cells, and when the cells age, the lysosomes are broken to release the hydrolytic enzymes, digest the whole cells and cause the cells to die. Since the microenvironment (such as polarity, pH, viscosity) of the cell is very different between lysosome and autophagosome, the microenvironment in autophagosomal formed by fusion of the two tends to change when autophagy occurs in the cell, and therefore, we can monitor the occurrence of autophagy by detecting the change of the microenvironment of the cell in the process.

The pH is an important influence factor of biochemical reaction balance in cells, and as an important property in a biological system microenvironment, the pH influences the operation of normal vital activity mechanisms of the cells and the smooth conversion of biomolecules. Abnormal changes in cellular pH are closely linked to various diseases and physiological processes in biological systems.

The two-photon fluorescent probe is used as a testing tool, when the property of a tested substance or the environment is changed, a fluorescent signal is correspondingly changed, the sensitivity is high, the operation is convenient, the stability is high, the photobleaching property is low, and the cell penetrability is high, so that the excellent performance is shown in the qualitative analysis of a cell layer. Thus, two-photon fluorescent probes can be selected to monitor the autophagy process by detecting changes in pH within the cytosol.

Disclosure of Invention

The invention aims to provide a two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells, and a preparation method and application thereof. According to the invention, a proper fluorescent probe structure is selected through molecular design so as to realize two-photon imaging qualitative detection of the pH change of the cell lysosome. The fluorescent probe has strong specificity and high sensitivity, and cytotoxicity experiments show that the fluorescent probe almost has no toxic or side effect on cells.

The invention relates to a two-photon pH ratio measurement fluorescent probe (Lyso-MPBC) for monitoring autophagy of cells, which is called a fluorescent probe for short, takes carbazole as a matrix, 4-methoxyphenylacetylene as an electron donating group, a lysosome positioning group as morpholine and benzimidazole as a pH response group, and has the following structural formula:

the invention discloses a preparation method of a two-photon pH ratio measurement fluorescent probe for monitoring autophagy of cells, which comprises the following steps:

adding compound A1g, 0.46g of o-phenylenediamine, 0.0082g of p-toluenesulfonic acid and 30mL of N, N-dimethylformamide into a three-neck flask, heating to 120 ℃ in an oil bath pot, and introducing argon for protection reaction for 12 hours; after the reaction is finished, spin-drying the liquid, extracting with dichloromethane and water, collecting the upper organic phase, and spin-drying to obtain a crude product; the crude product thus obtained was sampled and separated by column chromatography to obtain 0.7g (1.28mmoL) of the objective product in a yield of 60%.

The structural formula of the compound A is as follows:

the eluent used in the column chromatography column separation is obtained by mixing dichloromethane and methanol according to the volume ratio of 100: 1.

The synthesis process of the two-photon pH ratio metering fluorescent probe Lyso-MPCB for monitoring autophagy of the invention is as follows:

the invention discloses application of a two-photon pH ratio metering fluorescent probe Lyso-MPCB for monitoring autophagy of cells, which is used as a detection reagent when the pH of lysosomes in the cells is detected. The fluorescent probe (Lyso-MPCB) of the present invention can monitor the autophagy process of cells by detecting the change in lysosome pH.

Dissolving the fluorescent probe in DMSO to prepare 1mM mother liquor, taking 100 mu L of the mother liquor to be placed in a 10mL volumetric flask, and then adding solutions to be detected with different pH values (phosphoric acid-acetic acid-boric acid solution systems, adjusting to different pH values by adding 1mM hydrochloric acid or sodium hydroxide solution) to constant volume to prepare 10 mu M. The excitation wavelengths of the single photon and the two photon of the fluorescent probe are respectively 370nm and 760nm, and the change of the fluorescence spectrum in the wavelength range of 390-700nm is detected, so that the maximum fluorescence emission peak is red-shifted from 410nm to 475nm and is 65nm totally along with the reduction of the pH of the buffer solution from the alkalinity of 9.60 to the acidity of 3.20, and the red shift of the fluorescence intensity is enhanced.

The action mechanism of the fluorescent probe is that the tertiary nitrogen of the benzimidazole group in the fluorescent probe molecule contains a pair of lone electron pairs, the fluorescent probe molecule exists in different forms when the pH value of a test system is small and large, and when the probe molecule is excited by light to emit fluorescence, the excitation energy required for the electrons to transit from a ground state to an excited state is different, so that the obtained fluorescence spectrum is greatly changed. Lone-pair electrons in a low pH value system are combined with protons in the system to form an organic salt structure, at the moment, the electron-withdrawing capability of fluorescent probe molecules is enhanced, the performance of electron pushing and pulling is enhanced, the excitation energy required by the transition of ground state electrons to an excited state is reduced, and the fluorescence emission spectrum is subjected to transition to increase in fluorescence intensity; the lone electron pair cannot be combined with protons in a high pH value system, the conjugation of the whole fluorescent probe molecule is poor, the ability of pushing and pulling electrons is weakened, the excitation energy required for the ground state electrons to jump to an excited state is enhanced, and the fluorescence intensity is reduced.

The fluorescent probe has the advantages of simple structure, convenient synthesis, simple and convenient operation and sensitive reaction. The change of the pH of the microenvironment is detected by the change of the fluorescence intensity and the color, the toxicity to cells is low, and the method can be used for detecting the change of the pH in the cytolysosome and further monitoring the autophagy process of the cells.

The cell co-localization experiment proves that the probe can specifically target cell lysosomes, cytotoxicity test shows that the probe has little toxic or side effect on cells, and two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good permeability on MCF-7 cells, and meanwhile, the pKa of the fluorescent probe is calculated to be 3.88, so that the probe is very suitable for monitoring the pH variation range of the cell lysosomes, and the condition of the cell autophagy process can be monitored in real time by detecting the pH variation of the cell lysosomes.

Drawings

FIG. 1 shows UV absorption spectra of the inventive fluorescent probe Lyso-MPCB (10. mu.M) in buffer solutions of different pH values.

FIG. 2 is a graph showing fluorescence emission spectra of the inventive fluorescent probe Lyso-MPCB (10. mu.M) in buffer solutions of various pH values, each line being a test performed immediately after addition of the probe molecule.

FIG. 3 is a graph showing fluorescence emission patterns measured at pH 4.2 and pH 7.2 in a test solution prepared by back-and-forth adjustment of the fluorescent probe Lyso-MPCB of the present invention with 1mM hydrochloric acid and sodium hydroxide solution, respectively, and then calculating the ratio of fluorescence intensities at the fluorescence emission wavelengths of 475nm and 410nm (I)_475nm/I_410nm) The cycle chart of (1).

FIG. 4 shows two-photon absorption cross-sectional values of the fluorescent probe Lyso-MPCB (0.1mM) of the present invention under excitation of different wavelengths in buffer solutions of different pH values.

FIG. 5 shows the cell viability of the fluorescent probe Lyso-MPCB of the present invention after MCF-7 cells were cultured for 24 hours.

FIG. 6 is a photograph of imaging the localization of lysosomes in MCF-7 cells by the fluorescent probe Lyso-MPCB of the present invention. The probe Lyso-MPCB (10. mu.M) was added to MCF-7 cells and cultured for 30 minutes, and then the lysosomal dye LysoTracker Red FM (0.5. mu.M) was added thereto and the culture was continued for 10 minutes. Wherein the graph (a) shows the green channel (460-490nm), lambda _ex760 nm; (b) red channel (580-620nm), lambda_ex559 nm; (c) is a superimposed view of (a) and (b) channels; (d) is a scatter plot analysis of the fluorescence intensity corresponding to the (a) and (b) channels.

FIG. 7 shows the change in fluorescence after 4 hours of starvation-induced autophagy after 0.5 hour incubation of MCF-7 cells with the inventive fluorescent probe Lyso-MPCB molecule (10. mu.M). Wherein, the blue channel (390-420nm) is shown in graph (a 1-2); (b1-2) green channel (460-490 nm); (c1-2) is bright field; (d1) is a superimposed view of the (a1), (b1), and (c1) channels; (d2) is a superimposed view of the (a2), (b2), and (c2) channels.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

Example 1: synthesis of fluorescent probe molecule Lyso-MPCB

Adding compound A1g, 0.46g of o-phenylenediamine, 0.0082g of p-toluenesulfonic acid and 30mL of N, N-dimethylformamide into a three-neck flask, heating to 120 ℃ in an oil bath pot, and introducing argon for protection reaction for 12 hours; after the reaction is finished, spin-drying the liquid, extracting with dichloromethane and water, collecting the upper organic phase, and spin-drying to obtain a crude product; the crude product was sampled and separated by column chromatography to give the desired product 0.7g (1.28mmoL) with a yield of 60%. The structural formula of the compound A is as follows:

¹H NMR(400MHz,DMSO-d₆)12.84(s,1H),9.04(s,1H),8.41(s,1H),8.33(d,J＝8.6Hz,1H),7.84(d,J＝8.7Hz,1H),7.73(d,J＝8.5Hz,1H),7.65(d,J＝8.5Hz,2H),7.55(t,J＝11.5Hz,3H),7.20(dd,J＝5.5,2.6Hz,2H),7.02(d,J＝8.3Hz,2H),4.49(t,J＝7.0Hz,2H),3.82(s,3H),3.53(s,4H),2.30(s,6H),1.89-1.79(m,2H),1.53-1.47(m,2H).¹³C NMR(101MHz,DMSO-d₆)160.23,153.36,142.25,141.14,133.69,130.29,125.90,124.60,123.28,122.87,122.62,120.14,115.89,115.39,114.33,111.23,111.09,90.22,88.74,67.07,58.31,56.25,54.12,43.37,30.00,27.13.

example 2: fluorescence and two-photon testing of fluorescent probe molecules

Dissolving the fluorescent probe Lyso-MPCB in DMSO to prepare 1mM mother liquor, putting 100 mu L of the mother liquor in a 10mL volumetric flask, and then adding buffer solutions with different pH values (phosphoric acid-acetic acid-boric acid solution systems, adjusting to different pH values by adding 1mM hydrochloric acid or sodium hydroxide solution) to constant volume to prepare 10 mu M. Fluorescent probe single photon and double lightThe excitation wavelengths of the photons are 370nm and 760nm respectively, and the change of the fluorescence spectrum in the wavelength range of 390-700nm is detected. And passing through I corresponding to different pH values_475nm/I_410nmThe pKa value of the probe was calculated (3.88).

The fluorescence probe Lyso-MPCB was measured for fluorescence emission spectra at pH 4.2 and pH 7.2 in test solutions adjusted back and forth with 1mM hydrochloric acid and sodium hydroxide solution, respectively, and the ratio of fluorescence intensities at 475nm and 410nm was calculated (I)_475nm/I_410nm) The cycle chart (FIG. 3) shows that the number of cycles is 6.

The two-photon absorption cross section of the fluorescent probe (Lyso-MPCB) at pH 3.2 was measured using the two-photon measurement technique, and as can be seen from fig. 4, the maximum two-photon absorption cross section of the fluorescent probe molecule at pH 3.2 was 335GM, and the two-photon excitation wavelength was 760 nm.

Example 3: cytotoxicity test

MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide) assay was performed according to reported articles for cytotoxicity tests. The same batch of cells was added with 0, 5, 10, 15, 20. mu.M fluorescent probe, respectively, under the condition of 5% CO at 37 ℃₂For 24 hours, according to the formula of cell viability: percent cell survival ═ OD_{570 (sample)}/OD_{570 (control group)}× 100 (FIG. 5). As can be seen from FIG. 5, the cell viability is about 90% at a concentration of 20. mu.M, indicating that the fluorescent probes of the present invention are non-toxic to cells and can therefore be used to detect viscosity in cells.

Example 4: cell localization assay

MCF-7 cells were cultured in DEME (invitrogen) medium, and on the day before imaging, MCF-7 cells were placed in a confocal laser dish, and MCF-7 cells and 10. mu.M DMSO solution of a fluorescent probe Lyso-MPCB were imaged at 37 ℃ with 5% CO₂The cells were incubated in the incubator for 0.5 hour, washed 3 times with neutral PBS buffer solution, and then 0.5. mu.M of a commercial lysosomal stain LysoTracker Red FM solution was added to the petri dish for further incubation for 0.5 hour and washed 3 times with neutral PBS buffer solution. By pairsThe photon fluorescence confocal imaging is characterized in that a green channel tracker1 is arranged, the excitation wavelength is 760nm, the emission waveband is 460-490nm, and the channel is used for receiving the fluorescence emitted by the probe molecule Lyso-MPCB. A Red channel, tracker2, with an excitation wavelength of 559nm and an emission wavelength of 580-620nm was set up to receive fluorescence emitted by the commercial lysosomal stain Lysotracker Red FM (FIG. 6).

Example 5: autophagy monitoring

MCF-7 cells were cultured in DEME (invitrogen) medium, and on the day before imaging, MCF-7 cells were plated on flat-bottom surface dishes and imaged with MCF-7 cells and 10. mu.M DMSO solution of fluorescent probe Lyso-MPCB at 37 ℃ with 5% CO₂The cell culture chamber of (1) was incubated for 0.5 hour, and after washing well with a neutral PBS buffer solution or a culture solution, the medium was changed to HBSS (starvation medium for inducing autophagy of cells). Then, at the beginning (0h) and after a treatment period (4h), confocal imaging was performed with two-photon fluorescence, resulting in FIG. 7. Wherein, the blue channel (390-420nm) is shown in graph (a 1-2); (b1-2) green channel (460-490 nm); (c1-2) is bright field; (d1) is a superimposed view of the (a1), (b1), and (c1) channels; (d2) is a superimposed view of the (a2), (b2), and (c2) channels.

Claims

1. A two-photon pH ratiometric fluorescent probe for monitoring autophagy, comprising: carbazole is used as a matrix, 4-methoxyphenylacetylene is used as an electron donating group, a lysosome positioning group is morpholine, and benzimidazole is a pH response group;

the structural formula of the fluorescent probe is as follows:

2. use of the two-photon pH ratiometric fluorescent probe of claim 1 to monitor autophagy, wherein: the fluorescent probe is used for preparing a detection reagent for detecting the pH change of the autophagosomal in the cell.

3. Use of a two-photon pH ratiometric fluorescent probe for monitoring autophagy according to claim 1, characterized in that: the fluorescent probes are used to prepare detection reagents that monitor the extent of autophagy by detecting changes in the lysosome pH of cells.

4. Use according to claim 3, characterized in that it comprises the following steps:

dissolving the fluorescent probe in DMSO to prepare 1mM mother liquor, putting 100 mu L of the mother liquor in a 10mL volumetric flask, diluting to constant volume with the solution to be detected, and passing through a first detection channel: 390-420nm and a second channel: measurement change I of fluorescence spectrum peak ratio in wavelength range of 460-490nm_475nm/I_410nmThe pH change of the autophagy lysosome of the cells is quantitatively detected, so that the purpose of monitoring the autophagy process of the cells is achieved.