CN115160381A - Glutathione-responsive fluorescent probe, preparation method and application thereof - Google Patents
Glutathione-responsive fluorescent probe, preparation method and application thereof Download PDFInfo
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- CN115160381A CN115160381A CN202210994620.6A CN202210994620A CN115160381A CN 115160381 A CN115160381 A CN 115160381A CN 202210994620 A CN202210994620 A CN 202210994620A CN 115160381 A CN115160381 A CN 115160381A
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- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
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Abstract
The invention relates to the technical field of imaging of a fluorescent probe of a cyclometalated complex, in particular to a fluorescent probe responding to glutathione, a preparation method and application thereof. The complex is characterized by mass spectrum and nuclear magnetic resonance, the influence of glutathione and cysteine at different concentrations on the fluorescence intensity of the iridium complex is measured, and the influence of common interference factors such as amino acid, ions and pH in cells on the fluorescence of the iridium complex is eliminated. Cell imaging experiments show that the fluorescence intensity of the iridium complex in cells is enhanced along with the increase of incubation time, and the iridium complex is positioned in endoplasmic reticulum, so that the imaging and quantitative detection of glutathione in the cells are realized.
Description
Technical Field
The invention relates to the technical field of imaging of a fluorescent probe of a cyclometalated complex, in particular to a glutathione-responsive fluorescent probe, a preparation method and application thereof.
Background
Glutathione (GSH) is a tripeptide compound containing thiol, which is present in a very abundant amount in human cells, and mainly has antioxidant and detoxifying functions. Modern chemical and biological research shows that GSH plays an important role in aspects of matrix gene expression, metabolic regulation, immune function and the like in life activities, and particularly, the content change of the GSH in the processes of carcinogenesis and treatment is often related to the carcinogenesis degree and the treatment effect. Cisplatin chemotherapeutic drugs commonly used in clinical chemotherapy often generate drug resistance in the using process, wherein one reason is that high-concentration glutathione in cancer cells reacts with cisplatin to play a role in detoxifying and reduce the chemotherapeutic effect of the cisplatin. On the other hand, the change of GSH content in cancer cells is also related to cancer cell migration and apoptosis. Therefore, the detection and imaging of the glutathione content in the cells can help to understand the treatment of the tumor and the reason for the drug resistance.
At present, methods commonly used for detecting the content of glutathione comprise a plurality of methods, such as a colorimetric method, an iodometry method, an enzyme cycling method and the like, but the methods have advantages and defects. For example, the enzyme cycling method is often used for detecting the content of glutathione and oxidized glutathione in cells, the principle of detection is that firstly, cells are cracked to extract lysate, then oxidized glutathione reductase and NADPH are added into the lysate, oxidized glutathione can be continuously converted into reduced glutathione, TNB and glutathione are added for reaction under the condition that the reduced glutathione is not changed, and the content of all glutathione in the cells is measured by detecting the ultraviolet absorption of the TNB at 414nm, so that the method has the advantage of accurately quantifying the glutathione in the cells. However, the detection method is complicated, requires a large number of cells, requires cell lysis and multi-step pretreatment for pretreatment, and cannot dynamically monitor changes in glutathione content in living cells.
The fluorescence method is used as a non-invasive imaging means and is a method for detecting the content of glutathione, and compared with the method, the fluorescence method has the advantages of short reaction time, no need of complicated treatment on cells and capability of dynamically tracking the content change of the glutathione in living cells. At present, researches on glutathione detection by a fluorescence method mainly focus on organic micromolecular dyes, glutathione is detected by connecting an activating group such as an aldehyde group with dye molecules, but the reaction time and sensitivity of the aldehyde group to the glutathione are low, cysteine can also react with the aldehyde group, so that intracellular glutathione and cysteine cannot be well distinguished, in addition, some organic probes are troublesome to prepare, and fluorescence is easy to generate photobleaching, so that the stability of cell imaging is poor, a cyclometalated iridium complex has the advantages of good fluorescence stability, adjustable emission wavelength and the like, and is developed for detecting cysteine, hypochlorous acid and the like, but glutathione is detected at present, and almost no report for detecting glutathione is provided based on an azo binuclear iridium complex, so that the invention realizes that the response value of 0-0.5mM glutathione is close to 60 times changed by constructing the azo binuclear iridium complex through a one-step method, is positioned in an endoplasmic reticulum, and can realize specific detection of glutathione in the endoplasmic reticulum.
In view of the above-mentioned drawbacks, the inventors of the present invention have finally obtained the present invention through a long period of research and practice.
Disclosure of Invention
The invention aims to solve the problems that the existing glutathione fluorescent probe is troublesome to prepare, poor in selectivity, difficult to distinguish cysteine and sulfhydryl-containing substances, and poor in cell imaging stability due to photobleaching of fluorescence, and provides a glutathione specific response fluorescent probe, a preparation method and application thereof.
In order to achieve the purpose, the invention discloses a glutathione-responsive fluorescent probe, which is a binuclear iridium complex, wherein the chemical structural formula of the binuclear iridium complex is as follows:
the invention also discloses a preparation method of the glutathione-responsive fluorescent probe, which comprises the following specific steps: adding 4,4-azopyridine ligand into an iridium phenylpyridine precursor, adding silver trifluoromethanesulfonate into a mixed solvent of dichloromethane and methanol as a reaction solvent for reaction, filtering out silver chloride precipitate after the reaction is finished, spin-drying the reaction liquid, and purifying by column chromatography to obtain the binuclear iridium complex. Compared with the traditional method for preparing the iridium complex fluorescent probe, the method reduces one step, directly reacts the azo main ligand and the iridium precursor to obtain the iridium complex fluorescent probe, and saves time and solvent cost.
The molar ratio of 4,4-azopyridine ligand to phenylpyridine iridium precursor is 1:1.5.
the volume ratio of the dichloromethane to the methanol in the dichloromethane and methanol mixed solvent is 1:2.
the reaction temperature of the reaction is 65 ℃, and the reaction time is 48h.
The column chromatography purification adopts a silica gel column, and the eluent ratio is 50:2.
the invention also discloses application of the glutathione-responsive fluorescent probe in cancer cell glutathione imaging.
The recognition mechanism of the fluorescent probe of the invention is as follows:
the binuclear iridium complex is bridged through an azo group, N = N double bond in the azo group leads to the fluorescence quenching of the iridium complex, NH-NH is generated after the azo group is reduced by glutathione, the quenching effect disappears, and the fluorescence of the iridium complex is recovered, so that the detection of the content of the glutathione is realized. The detection mechanism of the invention is different from the detection mechanism of organic micromolecules, and mainly utilizes the reducibility of glutathione, but cysteine and other sulfhydryl proteins do not have reducibility, so that the probe cannot be interfered.
Compared with the prior art, the invention has the beneficial effects that:
1. the cyclic binuclear iridium complex is simple to synthesize, and a required probe can be obtained only by one step;
2. the binuclear iridium complex has high quantum yield, long fluorescence life and high light stability;
3. the fluorescence intensity of the metal iridium probe is gradually enhanced along with the increase of the concentration of glutathione, the change of the fluorescence intensity of the metal iridium probe to 0-0.5mM of glutathione is about 60 times, but the response to cysteine is only about 6 times, and the response is not obvious;
4. the cyclo-binuclear iridium complex has unobvious response to common amino acids and ions in cells under the same condition, can be well used for imaging glutathione in cancer cells, is positioned in an endoplasmic reticulum, and can realize detection of glutathione in the endoplasmic reticulum.
Drawings
FIG. 1 is a mass spectrum of a binuclear iridium complex;
FIG. 2 is a diagram of a binuclear iridium complex 1 H NMR chart;
FIG. 3 shows fluorescence intensities of binuclear iridium complexes at different concentrations of GSH;
FIG. 4 shows fluorescence intensities of binuclear iridium complexes at different concentrations of cysteine;
FIG. 5 shows fluorescence intensities of binuclear iridium complexes at different amino acids;
FIG. 6 shows fluorescence intensities of binuclear iridium complexes under different ions;
FIG. 7 shows fluorescence intensities of binuclear iridium complexes at different pH values;
FIG. 8 is a graph of fluorescence imaging of binuclear iridium complexes in MCF-7 cells;
FIG. 9 is a co-localization imaging diagram of binuclear iridium complexes in MCF-7 cells.
Detailed Description
The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.
1. Preparation of binuclear iridium complex
Weighing 4,4-azopyridine ligand of 100mg, adding 873mg phenylpyridine iridium precursor according to the molar ratio of 1.5, adding 50mL mixed solvent of dichloromethane and methanol (the volume ratio is 1:2), adding 417mg silver trifluoromethanesulfonate, heating and refluxing the reaction liquid for 48H, filtering out silver chloride precipitate after the reaction is finished, then spin-drying the reaction liquid, directly adopting silica gel column dry-method sampling, purifying by using a volume ratio of dichloromethane to methanol of 50 to obtain a reddish brown product, drying, collecting and storing for later use, and performing characterization by mass spectrometry as shown in figure 1 to show a divalent peak 685.22 of iridium, and simultaneously showing that the total H atom number is 48 in a nuclear magnetic hydrogen spectrum of figure 2, wherein the hydrogen atom number in the binuclear iridium complex is exactly 48, and the preparation of the binuclear iridium complex can be successfully determined by combining the mass spectrometry.
2. Fluorescence intensity change of binuclear iridium complex under different concentrations of GSH
Firstly weighing 2mg of binuclear iridium complex, dissolving the binuclear iridium complex by DMSO (dimethyl sulfoxide) to obtain a solution with the concentration of 10mM, weighing 5mg of GSH (glutathione) to be dissolved by water to prepare a mother solution with the concentration of 10mM, diluting the mother solution according to the concentration gradient to ensure that the final concentration of the solution is 20 mu M-1 mM and the concentration of iridium contained in the solution is 40 mu M, then wrapping the mother solution by tinfoil, incubating the mother solution in a water bath at 37 ℃ for 30min, and directly measuring the fluorescence intensity after the incubation is finished. When the fluorescence intensity curve is plotted, as shown in fig. 3, the fluorescence intensity of the binuclear iridium complex in a 0mM GSH solution is very low, about 180, the fluorescence intensity of iridium gradually increases with the increase of glutathione concentration, and when the glutathione concentration increases to 0.5mM, the fluorescence intensity of the iridium complex is 11000, which is increased by about 60 times. This shows that the iridium complex is capable of specifically detecting glutathione.
3. Fluorescence intensity change of binuclear iridium complex under different concentrations of Cys
Preparing a test solution: an iridium complex probe sample (mother liquor 10mM, dissolved by DMSO, the final concentration is 40 mu M) 2 mu L + Cys (mother liquor 10mM, dissolved by water, added with methanol (solvent) according to the concentration gradient (the final concentration is 20 mu M-1 mM), and prepared into 500 mu L of test solution, and added into a 0.6mL centrifuge tube to be mixed uniformly, wherein 8 groups of Cys test solutions with different concentrations are provided, and the Cys concentrations are respectively 0 (a control group, no. 1); 20 muM (experiment group, no. 2), 40 muM (experiment group, no. 3), 60 muM (experiment group, no. 4), 80 muM (experiment group, no. 5), 100 muM (experiment group, no. 6), 500 muM (experiment group, no. 7), 1mM (experiment group, no. 8), the experiment group is wrapped by tinfoil and incubated for 30min in water bath at 37 ℃, the fluorescence intensity is directly measured after the incubation is finished, and a fluorescence intensity curve is drawn.
4. Interference determination of cell main amino acid on iridium complex fluorescence intensity
The amino acid classes tested were: acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (tryptophan, proline, phenylalanine), basic amino acids (arginine, lysine), and GSH group and blank group as control.
Preparing a test solution: iridium complex probe sample (stock solution 10mM, dissolved in DMSO at a final concentration of 40. Mu.M) 2. Mu.L + different amino acids (stock solution 10mM, dissolved in buffer pH7.4 at a final concentration of 100. Mu.M) 5. Mu.L + methanol (solvent) was prepared as 500. Mu.L of test solution, which was added to a 0.6mL centrifuge tube and mixed well. There are 9 groups of test solutions, which are: no amino acid was added (blank, no. 1); adding amino acids (2-8) with different acid and base; GSH (No. 9) was added. And then, the experimental group is wrapped by tinfoil and incubated for 30min in water bath at 37 ℃, the fluorescence intensity of the experimental group is directly measured after the incubation is finished, and a fluorescence intensity curve is drawn. As shown in FIG. 5, it can be seen that the fluorescence of the binuclear iridium complex is slightly affected by the common amino acids in cells, indicating that the detection of glutathione by the binuclear iridium complex in cells is not affected by the amino acids, which has not been considered in the prior art.
5. Interference determination of main ions in cells on fluorescence intensity of iridium complex
Preparing an ionic solution: the experimental raw material is Na 2 CO 3 ,NaHCO 3 ,Na 2 SO 4 ,NaCl,ZnCl 2 . The purity was AR grade. The concentration of the mother solution of the prepared ionic solution is 10mM, and ultrapure water is used as a solvent. Preparing a test solution: iridium complex probe sample (mother liquor 10mM, using DMS)O dissolution, 40. Mu.M final concentration) 2. Mu.L + ion solution (mother liquor 10mM, dissolved with water, 100. Mu.M final concentration) 5. Mu.L + methanol (solvent) to prepare 500. Mu.L of test solution, which was added to a 0.6mL centrifuge tube and mixed well. Wherein the control group did not contain ionic solution. There are 6 groups of test solutions, which are: no ionic solution was added, GSH (control, no. 1) was added; CO 2 3 2- (No. 2); HCO 3 - (No. 3), SO 4 2- (No. 4), cl - (No. 5), zn 2+ (No. 6). And then, the experimental group is wrapped by tinfoil and incubated for 30min in water bath at 37 ℃, the fluorescence intensity of the experimental group is directly measured after the incubation is finished, and a fluorescence intensity curve is drawn. As shown in FIG. 6, the fluorescence of the binuclear iridium complex is not affected by anions and cations commonly found in cells, and the influence of the anions on the fluorescence intensity of the iridium is almost negligible, so that the iridium complex can be further ensured to be used for detecting the content of glutathione in the cells.
6. Change of fluorescence property of iridium complex under different pH conditions
Buffer solution with certain pH gradient is prepared: disodium hydrogen phosphate dodecahydrate (Na) 2 HPO 4 ·12H 2 O) and citric acid (C) 6 H 8 O 7 ) Preparing 6 groups of buffer solutions with different pH values, wherein the pH values are 5.0,5.4,5.8,6.2,6.6,7.0,7.4 respectively. Preparing a test solution: iridium complex probe sample (mother solution 10mM, dissolved in DMSO, final concentration 40 μ M) 2 μ L + buffer-methanol system solvent (buffer volume: methanol volume = 1:1) was prepared as 500 μ L test solution, which was added to a 0.6mL centrifuge tube and mixed well. The total number of 7 groups of test solutions with different pH values is as follows: 5.0 (number 1); 5.4 (No. 2); 5.8 (No. 3); 6.2 (No. 4); 6.6 (No. 5); 7.0 (No. 6); 7.4 (No. 7). And then, the experimental group is wrapped by tinfoil and incubated for 30min in water bath at 37 ℃, the fluorescence intensity of the experimental group is directly measured after the incubation is finished, and a fluorescence intensity curve is drawn. The result is shown in fig. 7, the fluorescence intensity of the binuclear iridium complex is not changed in solutions with different pH values, which indicates that the fluorescence intensity of the binuclear iridium complex is not influenced by the pH value, further ensures that the detection of glutathione by the binuclear iridium complex in cells is not influenced by the pH value, and ensures that the iridium complex can specifically detect glutathione in cellsA glycylglycine.
7. Cellular uptake imaging
And taking out a part of the passaged human breast cancer cells from a constant temperature incubator at 37 ℃, and placing the cells under a microscope to observe the cell state, wherein the growth area of the adherent cells is 1/2 of the visual field. Adding 10 mu L of biological DMSO into an empty centrifuge tube for assisting dissolution, adding 8 mu L of iridium complex probe mother liquor into the centrifuge tube, sucking a proper amount of liquid from 2mL of culture medium, adding the liquid into the centrifuge tube, blowing and uniformly mixing the liquid by using a pipette gun, and uniformly adding the liquid into a cell culture medium. The final concentration of iridium complex probe in the medium was 20. Mu.M. After the medicine is added, the culture dish is rotated and shaken to be mixed evenly, the outside of the culture dish is wiped by alcohol cotton, and then the culture dish is put into an incubator at 37 ℃ for continuous culture. Repeating the operation of the step 3 every two hours, wherein 4 cells are divided into 4 groups for cell uptake imaging, and the groups are cultured for 8 hours, 6 hours, 4 hours and 2 hours respectively. After the 4 groups of cells were cultured for 8h, 6h, 4h and 2h, respectively, they were taken out from the 37 ℃ incubator. The original medium in the 4 dishes was aspirated, and the cells were washed twice with 2mL PBS buffer added along the walls, followed by 1mL PBS. The cellular uptake imaging was then observed with a confocal laser microscope. Excitation at 405nm and reception at 520-540nm was used. The result is shown in fig. 8, it can be seen from fig. 8 that because the cell is a cancer cell, the glutathione concentration of the cell itself is high, the binuclear iridium complex has significant fluorescence in the cell, and after the drug is added for 4 hours, the significant fluorescence can be seen, which indicates that the iridium complex enters the cell rapidly, which is beneficial to rapidly and conveniently detecting the glutathione in the cell. After the medicine is added for 8 hours, the iridium generates obvious fluorescence in cells, and the cells can be clearly observed.
8. Cell co-localization imaging
Adding 10 mu L of biological DMSO for assisting dissolution into an empty centrifuge tube, adding 8 mu L of iridium complex probe mother liquor into the empty centrifuge tube, sucking a proper amount of liquid from 2mL of culture medium, adding the liquid into the centrifuge tube, blowing and beating the liquid by using a pipette, and uniformly adding the liquid into a cell culture medium. The final concentration of iridium complex probe in the medium was 20. Mu.M. After the medicine is added, the culture dish is rotated and shaken to be mixed evenly, and the culture dish is wiped by alcohol cotton and then is put into an incubator at 37 ℃ for continuous culture. Repeat 3 groups at the same time, the operation is the same. After 3 groups of cells are cultured for 8h at 37 ℃,1 mu L of each lipid drop LDs probe, mitochondria MTG probe and endoplasmic reticulum ER probe are respectively added into the three, 10 mu L of biological DMSO is used for assisting dissolution, and the three are continuously put into a 37 ℃ incubator for culture for 0.5h. And then, observing the co-localization imaging of the organelle probe and the iridium complex probe by using a laser confocal microscope. The imaging of the three organelle probes is realized by 488nm excitation and 500-520nm receiving. The iridium complex probe is as above. As shown in FIG. 9, it can be seen that the iridium complex fluorescence and the endoplasmic reticulum probe have better coincidence degree, but do not have better coincidence with mitochondria and lipid droplets, which indicates that the iridium complex is positioned in the endoplasmic reticulum of the cell, and the glutathione in the endoplasmic reticulum can be detected. This function is not achieved in the prior invention patents.
The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. It will be appreciated by those skilled in the art that many variations, modifications, and equivalents may be made thereto without departing from the spirit and scope of the invention as defined in the claims.
Claims (7)
2. the preparation method of the glutathione-responsive fluorescent probe as claimed in claim 1, characterized by comprising the following specific steps: adding 4,4-azopyridine ligand into a phenylpyridine iridium precursor, adopting a mixed solvent of dichloromethane and methanol as a reaction solvent, adding silver trifluoromethanesulfonate for reaction, filtering out silver chloride precipitate after the reaction is finished, spin-drying the reaction liquid, and purifying by column chromatography to obtain the binuclear iridium complex.
3. The method for preparing a glutathione-responsive fluorescent probe as claimed in claim 2, wherein the molar ratio of 4,4-azopyridine ligand to phenylpyridine iridium precursor is 1:1.5.
4. the method for preparing a glutathione-responsive fluorescent probe as claimed in claim 2, wherein the volume ratio of the dichloromethane and the methanol in the mixed solvent of the dichloromethane and the methanol is 1:2.
5. the method for preparing a glutathione-responsive fluorescent probe as claimed in claim 2, wherein the reaction temperature is 65 ℃ and the reaction time is 48h.
6. The method for preparing a glutathione-responsive fluorescent probe as claimed in claim 2, wherein the column chromatography purification adopts a silica gel column, and the eluent ratio is dichloromethane to methanol 50:2.
7. use of the glutathione-responsive fluorescent probe of claim 1 for glutathione imaging of cancer cells.
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