阅读说明：本技术 一种溶酶体靶向荧光探针及其制备方法和应用 (Lysosome targeted fluorescent probe and preparation method and application thereof ) 是由 王慧 胡磊 汪明 陈曦 于坤 许丙嵩 于 2020-12-24 设计创作，主要内容包括：本发明公开了一种溶酶体靶向荧光探针及其制备方法和应用,以具有强给电子能力的三苯胺单醛衍生物与强吸电子能力的2-甲基喹啉碘盐反应,简洁高效地制备了一种具有D-π-A构型的三苯胺类衍生物溶酶体靶向荧光探针,其最大发射峰在646nm左右,且随着粘度的增加,其荧光强度显著增强；激光共聚焦显微成像结果显示,本发明制备的溶酶体靶向荧光探针能够穿透细胞膜,靶向在细胞内的溶酶体部位,由于探针具有较好的光稳定性,其能够实时监测溶酶体的粘度变化、及融合和迁移的过程。(The invention discloses a lysosome targeted fluorescent probe and a preparation method and application thereof, wherein a triphenylamine single aldehyde derivative with strong electron donating capability reacts with 2-methylquinoline iodonium salt with strong electron withdrawing capability to simply and efficiently prepare the triphenylamine derivative lysosome targeted fluorescent probe with a D-pi-A configuration, the maximum emission peak is about 646nm, and the fluorescence intensity is obviously enhanced along with the increase of viscosity; laser confocal microscopy imaging results show that the lysosome targeted fluorescent probe prepared by the invention can penetrate cell membranes and target lysosome parts in cells, and the probe has better light stability, so that the viscosity change of lysosomes and the fusion and migration processes can be monitored in real time.)

1. A lysosomal targeting fluorescent probe having the structural formula:

2. the method of making a lysosomal targeting fluorescent probe of claim 1, comprising the steps of: dissolving a compound M1 and a compound M2 in an organic solvent, carrying out reflux reaction by using organic base as a catalyst, cooling to room temperature after the reaction is finished, and then carrying out suction filtration, washing and drying to prepare the lysosome targeted fluorescent probe;

the structural formula of the compound M1 is as follows:

the structural formula of the compound M2 is as follows:

3. the production method according to claim 2, wherein the organic solvent is ethanol; the organic base is piperidine.

4. The method according to claim 2, wherein the ratio of the amounts of the substance of compound M1, compound M2, and catalyst is 1: 1.1-1.2: 0.6 to 0.7.

5. The method according to claim 2, wherein the concentration of the compound M1 relative to the organic solvent is 0.05-0.1M.

6. The preparation method according to claim 2, wherein the time of the reflux reaction is 22-26 h.

7. Use of the lysosomal targeting fluorescent probe of claim 1 for lysosomal labeling or monitoring of lysosomal viscosity changes.

8. Use of the lysosomal targeting fluorescent probe of claim 1 for real-time monitoring of lysosomal fusion and migration processes.

9. A method for lysosomal labeling using the lysosomal targeting fluorescent probe of claim 1, comprising the steps of: and (3) co-culturing the biological cells and the lysosome targeted fluorescent probe for 20 minutes, and developing by using a laser confocal microscope.

Technical Field

The invention belongs to the technical field of molecular probes, and particularly relates to a lysosome targeted fluorescent probe and a preparation method and application thereof.

Background

In a living body, intracellular viscosity plays an important role in controlling information transmission, substance transport, and interaction between biological macromolecules. Abnormalities in intracellular viscosity are closely related to cellular dysfunction and many diseases such as alzheimer's disease, arteriosclerosis, cellular malignancies, and diabetes. Lysosomes are almost all animal cells, are vesicular organelles surrounded by a single-layer membrane and containing various acid hydrolases, and play important roles in the aspects of intracellular digestion, maintenance of normal metabolic activities of cells, defense and the like. Lysosomes, which are the digestive organs of cells, are accompanied by changes in viscosity during the process of uptake and degradation of biological macromolecules. In addition, if a lysosome is deficient in a hydrolase due to a genetic defect, the corresponding substrate cannot be degraded and accumulated in the lysosome, which may overload the lysosome to cause lysosomal storage diseases. Therefore, monitoring changes in viscosity in lysosomes has important application value.

In recent years, laser confocal fluorescence microscopy has become an indispensable research tool for research in the biomedical field. Compared with the common fluorescence microscopic imaging technology, the laser confocal fluorescence microscopic imaging technology has the advantages that: high spatial resolution and selectivity, small signal interference, etc., provide a more sharp tool for visualizing and analyzing dynamic processes of living cells and tissues. However, the emission wavelength of most of the conventional lysosome viscosity fluorescent probes is short (<600nm), and the probes are easily interfered by the autofluorescence of blue-green areas in cells, have low tissue penetration depth and are not beneficial to biological imaging.

Disclosure of Invention

In order to solve the technical problems, the invention provides a lysosome targeted fluorescent probe, the maximum emission peak of which is about 646nm, which can eliminate the interference of autofluorescence in organisms, thereby reducing background noise, improving the signal-to-noise ratio, and having smaller light damage and stronger permeability.

The invention also provides a preparation method of the lysosome targeted fluorescent probe, and the preparation method has the advantages of easily available raw materials, low cost, mild reaction conditions, simple post-treatment and higher yield, and makes commercialization of the lysosome targeted fluorescent probe possible.

The invention also provides the application of the lysosome targeted fluorescent probe in lysosome marking or monitoring lysosome viscosity change or monitoring the fusion and migration process of the lysosome in real time, wherein the lysosome targeted fluorescent probe can penetrate through cell membranes, target lysosome parts in cells and track the fusion and migration process of the lysosome; and as the viscosity of the lysosome increases, the fluorescence intensity increases.

The invention also provides a method for labeling lysosomes by using the lysosome targeted fluorescent probe, biological cells and the lysosome targeted fluorescent probe are cultured for 20 minutes together, and a laser confocal microscope is used for developing.

In order to achieve the purpose, the invention adopts the following specific technical scheme:

a lysosomal targeting fluorescent probe having the structural formula:

the preparation method of the lysosome targeted fluorescent probe provided by the invention comprises the following steps: dissolving a compound M1 and a compound M2 in an organic solvent, carrying out reflux reaction by using organic base as a catalyst, cooling to room temperature after the reaction is finished, and then carrying out suction filtration, washing and drying to prepare the lysosome targeted fluorescent probe;

the structural formula of the compound M1 is as follows:

the knot of the Compound M2The structure formula is as follows:

further, the organic solvent is ethanol; the organic base is piperidine.

The mass ratio of the compound M1 to the compound M2 to the catalyst is 1: 1.1-1.2: 0.6-0.7, preferably 1: 1.13: 0.67.

the concentration of the compound M1 relative to the organic solvent is 0.05-0.1M, and preferably 0.06M.

The time of the reflux reaction is 22-26 h, preferably 24 h.

According to the invention, the triphenylamine monoaldehyde derivative with strong electron donating capability reacts with the 2-methylquinoline iodonium salt with strong electron withdrawing capability to simply and efficiently prepare the triphenylamine derivative lysosome targeted fluorescent probe with the D-pi-A configuration, the maximum emission peak of the probe is about 646nm, and the fluorescence intensity of the probe is remarkably enhanced along with the increase of viscosity. Laser confocal microscopy imaging results show that the lysosome targeted fluorescent probe prepared by the invention can penetrate cell membranes and is targeted to a lysosome part in cells. Because the probe has better light stability, the probe can monitor the process of lysosome fusion and migration in real time.

The invention has the following beneficial effects:

1. the lysosome targeted fluorescent probe provided by the invention has the advantages of easily available synthetic raw materials, low cost, mild reaction conditions, simple post-treatment and higher yield, and can be commercialized.

2. The emission wavelength of the lysosome targeted fluorescent probe provided by the invention is about 646nm, so that the biological background can be effectively reduced, and the signal to noise ratio of biological imaging is improved.

3. The fluorescence intensity of the lysosome targeting fluorescent probe provided by the invention is obviously enhanced along with the increase of viscosity, and the fluorescence intensity can be enhanced to 458 times.

4. The lysosome targeted fluorescent probe provided by the invention is a fluorescent probe which can target lysosomes in cells and can monitor the change of lysosome viscosity.

5. The lysosome targeted fluorescent probe provided by the invention can track the process of lysosome fusion and migration in real time.

6. Compared with a commercial lysosome probe, the lysosome targeted fluorescent probe has larger Stokes shift, the excitation wavelength is 514nm, the emission wavelength is 646nm, and the Stokes shift is 132nm, so that the background interference can be reduced.

Drawings

FIG. 1 is a scheme of the synthesis of a lysosomal targeting fluorescent probe;

FIG. 2 is a nuclear magnetic hydrogen spectrum of the lysosomal targeting fluorescent probe prepared in example 1;

FIG. 3 is an infrared spectrum of the lysosomal targeting fluorescent probe prepared in example 1;

FIG. 4 is a mass spectrum of the lysosomal targeting fluorescent probe prepared in example 1;

FIG. 5 is the fluorescence emission spectra (A) of the lysosomal targeting fluorescent probe prepared in example 1 in different volume fractions of water/glycerol viscous medium; fluorescence histogram after interaction of compound L with amino acids, BSA, DNA, RNA, wherein: l-phenylalanine, 2. L-alanine, 3.BSA, 4. L-cysteine, 5.DNA, 6.GSH, 7. L-glutamic acid, 8. L-histidine, 9. L-leucine, 10. L-proline, 11.RNA, 12. L-serine, 13. L-threonine, 14. L-valine, 15. L-arginine, 16, L-tyrosine, 17.99% glycerol (B);

FIG. 6 shows the results of co-localization of human hepatoma cells with the lysosomal targeting fluorescent probe prepared in example 1 and commercial dyes with different targeting functions: wherein, panels A1-A4 are development diagrams of compound L after interaction with human hepatoma cells; FIGS. B1-B4 correspond to the results of visualization after co-incubation of human hepatoma cells after interaction of Lysotracker green (lysosome commercial stain), Mitotracker green (mitochondrial commercial stain), Hoechst 33342 (nuclear commercial stain), LCS LipidOX Deep (lipid droplet commercial stain), respectively, with Compound L; FIGS. C1-C4 correspond to the superimposed results of FIGS. A1-A4 and FIGS. B1-B4, respectively; plots D1-D4 correspond to Pearson's coefficient values, respectively, for co-localization results;

FIG. 7 is an image of cells after 20 minutes of co-incubation of the lysosomal targeted fluorescent probe prepared in example 1 with human hepatoma cells, without the addition of (A) and with the addition of (B) dexamethasone;

FIG. 8 is an image of cells after 20 minutes of co-incubation of the lysosomal targeted fluorescent probe prepared in example 1 with human hepatoma cells and 30 minutes of incubation with chloroquine solution.

Detailed Description

The present invention will be described in detail with reference to examples.

The synthesis method of the compound M1 used in the present invention is described in CrystEngComm,2017,19: 6489-; synthetic methods for compound M2 reference angelw.chem.int.ed., 2018,57: 16506-16510.

Example 1

A lysosomal targeting fluorescent probe (compound L) of the formula:

the synthetic route of the lysosome targeting fluorescent probe is shown in figure 1, and the preparation method comprises the following steps:

m1(0.43g,1.5mmol), M2(0.48g,1.7mmol) and 25mL of ethanol were added in this order to a 100mL round-bottom flask, 100. mu.L of piperidine was used as a catalyst, and the mixture was refluxed for 24 hours, cooled to room temperature after the reaction was completed, and a solid precipitated, and then the filtrate was filtered under reduced pressure, and the filter cake was washed with ethanol 3 times and dried under vacuum for 12 hours to obtain a violet-black solid 0.68g, with a yield of 81.9%. And the infrared spectrum, the hydrogen spectrum and the mass spectrum are characterized, and the results are as follows:

¹H NMR(400MHz,d₆-DMSO) delta 8.45-8.40(d,1H),8.26-8.02(m,4H),7.94-7.88(m,1H),7.76-7.63(m,2H),7.49-7.33(m,5H),7.16-7.10(m,6H),6.13-6.01(t,2H),4.15(s, 3H); as shown in fig. 2, H on the phenolic hydroxyl group in the structural formula showed no peak in the hydrogen spectrum due to the comparison of the active waves.

IR(KBr,cm^-1) selected bands:3451.2,3057.0,1608.6,1584.5,1486.1,1437.3,1329.7,1291.1,1273.1,1227.5,1165.1,1133.2,1110.2,987.7,821.2,752.7,693.8; as shown in fig. 3.

ESI-MS:429.2014([M-I]⁺) (ii) a As shown in fig. 4.

The compound L prepared in this example was dissolved in dimethyl sulfoxide solvent to prepare 10^-3Absorbing 50 mu L of mother liquor, respectively dispersing in 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 99% of water/glycerol viscous media with different volume fractions, wherein the total volume of each system is 5mL, the final concentration of the compound L is 10 mu M, and testing the fluorescence emission spectrum of the compound L at an excitation wavelength of 514 nm; as a result, as shown in FIG. 5A, it can be seen that the fluorescence intensity of Compound L gradually increased with the increase in the viscosity of the medium.

The compound L prepared in this example was dissolved in dimethyl sulfoxide solvent to prepare 10^-3Absorbing 50 mu L of mother liquor, respectively dispersing in PBS buffer solution, 99% glycerol solution and PBS buffer solution containing amino acid, BSA, DNA and RNA, wherein the total volume of each system is 5mL, the final concentration of the compound L in each system is 10 mu M, the final concentration of the amino acid, BSA, DNA and RNA is 200 mu M, testing the fluorescence emission spectrum under the excitation wavelength of 514nm, and taking a dispersion medium as a horizontal coordinate, I/I₀Plotted as the ordinate, where I is the fluorescence intensity value at 646nm after the action of Compound L with different analytes, I₀Fluorescence intensity value at 646nm for compound L in PBS buffer; the results are shown in fig. 5B, from which it can be seen that compound L exhibits strong fluorescence only in viscous medium, and that the fluorescence emission of compound L in viscous medium can be seen without interference from other analytes.

Example 2

Lysosomal targeting studies with lysosomal targeting fluorescent probes

Human hepatoma cells (HepG2 cell) were seeded on a laser confocal dish (NEST cat # 801002) 10 cells per well⁶The cells can be used when the cell density is increased to 60%. Compound L, prepared in example 1, was formulated into 10 using dimethylsulfoxide as solvent^-2The mother solution was diluted to 8. mu.M with the culture medium, and 1mL of the mother solution was added to each well-cultured human liver cancer cells (HepG2 cell) in a medium containing 95% air and 5% CO₂Gas incubator at 37 deg.CActing for 20 minutes, washing for 2 times by using PBS buffer solution, observing the result on a Leica TCS SP8 laser confocal microscope device, and setting the excitation wavelength to be 514 nm; as shown in fig. 6, it can be seen that compound L is able to target the lysosomal site within the cell.

To demonstrate that the probes of the present invention were able to target lysosomes, four groups of human liver cancer cells (HepG2 cells) were separately treated with 8. mu.M Compound L in a solution containing 95% air and 5% CO as described above₂The reaction was carried out in an air incubator at 37 ℃ for 20 minutes, washed with PBS buffer solution 2 times, and then subjected to confocal laser imaging at an excitation wavelength of 514nm, the results of which are shown in FIG. 6A 1-A4;

then co-incubated with Lysotracker green, Mitotracker green, Hoechst 33342 and LCS LipidOX Deep Red for 20 minutes, respectively, washed 2 times with PBS buffer solution, and laser confocal development was performed at 488nm, 405nm and 633nm excitation wavelengths, respectively, as shown in FIG. 6B 1-B4; FIGS. C1-C4 correspond to the superimposed results of FIGS. A1-A4 and FIGS. B1-B4, respectively; plots D1-D4 correspond to Pearson coefficient values for co-localization results, respectively. From figure 6 it can be demonstrated that compound L is able to target lysosomes within cells.

Example 3

Lysosomal targeted fluorescent probes for monitoring changes in viscosity in lysosomes

To further investigate the change in fluorescence intensity of the lysosomal targeting fluorescent probe after external environmental stimuli affected the in vivo viscosity of lysosomes, this example selected dexamethasone, an immunosuppressant that could act as a membrane of the lysosomal cell membrane, which induced an increase in lysosomal viscosity. Two groups of human liver cancer cells (HepG2 cell) were mixed with 8. mu.M of Compound L according to the method of example 2 in the presence of 95% air and 5% CO₂The culture medium is washed with PBS buffer solution for 2 times, fresh culture medium is added, 5 μ M dexamethasone is added into one group of human hepatoma cells, and the mixture is added into a culture medium containing 95% air and 5% CO₂Acting in an incubator with gas at 37 ℃ for 30 minutes, and then carrying out laser confocal development at an excitation wavelength of 514nm, wherein the excitation wavelength is set to be 514 nm; as shown in FIG. 7, from which can be seenIt is shown that when dexamethasone is used to stimulate cells, the fluorescence intensity of the probe is significantly enhanced, which indicates that the change of the in vivo viscosity of the lysosome can induce the change of the spatial configuration of the probe L, thereby showing bright red fluorescence.

Example 4

Lysosome target fluorescent probe tracking process of lysosome fusion and migration

Human hepatoma cells (HepG2 cell) were mixed with 8. mu.M Compound L according to the method of example 2 in a mixture containing 95% air and 5% CO₂The culture medium is washed with PBS buffer solution for 2 times, fresh culture medium is added, and 5 μ M chloroquine solution is added, wherein the chloroquine solution is used for inducing lysosome movement under the condition of keeping cells healthy, and the lysosome movement is induced in the presence of 95% air and 5% CO₂Acting in an incubator with gas at 37 ℃ for 30 minutes, and then carrying out laser confocal development at an excitation wavelength of 514nm, wherein the excitation wavelength is set to be 514 nm; as shown in fig. 8, it can be seen that probe L can monitor the process of lysosomal fusion and migration in real time.

The above detailed description of a lysosomal targeting fluorescent probe and its method of preparation and use with reference to the examples is illustrative and not restrictive, and several examples can be cited within the scope of the invention, and thus variations and modifications that do not depart from the general concept of the invention are intended to be within the scope of the invention.