Fixable lysosome fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a fixable lysosome fluorescent probe as well as a preparation method and application thereof. The lysosome fluorescent probe is a compound with a structure as shown in a formula (I), in the formula (I), R1 is any one of hydrogen and C1-C5 n-alkyl; r2 is any one of hydrogen and alkoxy with 1 to 3 carbon atoms; and X is any one of a halogen atom, BF4 and ClO4. The lysosome fluorescent probe does not depend on the acidity of the lysosome, and can image and trace the lysosome when the lysosome is alkalized.

Description

Technical Field

[0001] This invention relates to the field of probes, and more particularly to an immobilizable lysosomal fluorescent probe that is independent of acidic environments, its preparation method, and its applications. Background Technology

[0002] Lysosomes are the "digestive workshops" within cells, participating in the recycling and reuse of substances. They are important organelles with a variety of key biological functions. Lysosomes play a multifaceted and crucial role in cellular physiology and pathology, from the degradation and recycling of intracellular substances to cell signaling and immune responses. Their function is essential for maintaining cellular homeostasis and health. Furthermore, lysosomal dysfunction is closely related to various diseases and plays an important role in drug delivery and treatment. Therefore, in-depth research into the biological functions of lysosomes and their mechanisms of action in diseases is of great significance for developing new therapeutic strategies and drugs. Lysosomal fluorescence imaging is an important tool in lysosomal research; therefore, lysosomal fluorescent probes are widely used in medical, life science research, and clinical diagnostics.

[0003] Currently available lysosomal fluorescent probes are located in lysosomes in response to acidity. They can only be used for staining and imaging acidic lysosomes in live cells, but cannot be used to stain and image tracing alkalized lysosomes during physiological and pathological processes or lysosomes in fixed cells. Summary of the Invention

[0004] To address the problem that lysosomal fluorescent probes in the prior art depend on the acidity of lysosomes, this invention provides an immobilizable lysosomal fluorescent probe, its preparation method, and its application.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides an immobilizable lysosomal fluorescent probe, wherein the lysosomal fluorescent probe is a compound having the structure shown in formula (I):

[0006] Among them, R 1 R is any one of hydrogen and C1-C5 n-alkyl groups; 2 X is any one of hydrogen and C1~C3 alkoxy groups; X is any one of halogen atoms, BF4 and ClO4.

[0007] According to some embodiments of the present invention, the halogen atom is any one of iodine, bromine and chlorine.

[0008] According to some embodiments of the present invention, the R 1 In this context, the C1-C5 n-alkyl group is any one of methyl, ethyl, n-propyl, n-butyl, and n-pentyl.

[0009] According to some embodiments of the present invention, the R 2 In this context, the alkoxy group of C1 to C3 is any one of methoxy, ethoxy, and propoxy.

[0010] According to some preferred embodiments of the present invention, the lysosomal fluorescent probe is (E) -4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide or (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

[0011] In this invention, R 1 For methyl, R 2 When X is hydrogen and iodine, the resulting immobilizable lysosomal fluorescent probe is: (E) -4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

[0012] In this invention, R 1 For methyl, R 2 When X is methoxy and iodine, the resulting immobilizable lysosomal fluorescent probe is: (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

[0013] Secondly, the present invention provides a method for preparing an immobilizable lysosomal fluorescent probe, the method comprising the following steps: S1. Place 4-methylquinoline (formula (II)) and the substituted long-chain alkane shown in formula (III) in a solvent and heat to reflux to generate the intermediate shown in formula (IV); S2. The intermediate, the compound shown in formula (V) and the catalyst are mixed and heated under reflux. After reflux, impurities are removed to obtain the immobilizable lysosomal fluorescent probe; wherein the catalyst includes piperidine. .

[0014] According to some embodiments of the present invention, the solvent includes ethanol.

[0015] According to some embodiments of the present invention, the molar ratio of the compound represented by formula (V) to 4-methylquinoline is 1:(1.0~2.0).

[0016] According to some embodiments of the present invention, in step S1, the temperature of the reflux reaction is 80~90°C and the time is 1~5 days; in step S2, the temperature of the reflux reaction is 80~90°C and the time is 1~5 days.

[0017] According to some preferred embodiments of the present invention, the compound represented by formula (V) is indole-3-carboxaldehyde; the substituted long-chain alkane is 1-iodohexadecane. The preparation method of the lysosomal fluorescent probe using indole-3-carboxaldehyde and 1-iodohexadecane as reactants is as follows: S01. Prepare an ethanolic mixed solution of 4-methylquinoline and 1-iodohexadecane; S02. The ethanol mixture from step S01 is refluxed under heating and stirring conditions; S03. After the reflux reaction in step S02 is completed, add an ethanol solution of indole-3-carboxaldehyde; S04. Add the catalyst piperidine to the mixed solution obtained in step S03, heat to carry out reflux reaction, and after the reflux reaction is completed, slowly cool to room temperature, evaporate excess solvent, and obtain the organic solid product to be purified. S05. The organic solid product to be purified is subjected to column chromatography, using a dichloromethane / methanol mixed solvent as the eluent. After drying, a red powder is obtained. The red powder is a fixable lysosomal fluorescent probe. (E) -4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

[0018] According to some preferred embodiments of the present invention, the compound represented by formula (V) is 5-methoxy-3-formylindole; the substituted long-chain alkane is 1-iodohexadecane. The preparation method of the lysosomal fluorescent probe using 5-methoxy-3-formylindole and 1-iodohexadecane as reactants is as follows: S001. Prepare an ethanolic mixed solution of 4-methylquinoline and 1-iodohexadecane; S002. The ethanol mixture solution from step S001 is subjected to reflux reaction under heating and stirring conditions; S003. After the reflux reaction in step S002 is completed, add an ethanol solution of 5-methoxy-3-formylindole; S004. Add the catalyst piperidine to the mixed solution obtained in step S003, heat to carry out reflux reaction, and after the reflux reaction is completed, slowly cool to room temperature, evaporate excess solvent, and obtain the organic solid product to be purified. S005. The organic solid product to be purified is subjected to column chromatography, using a dichloromethane / methanol mixed solvent as the eluent. After drying, a dark red powder is obtained. The dark red powder is a fixable lysosomal fluorescent probe. (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

[0019] Thirdly, the present invention provides an application of the lysosomal fluorescent probe described herein in any of the aspects A1) to A4): A1) Marking and / or locating lysosomes in cells for purposes other than disease treatment and diagnosis; A2) Monitoring of lysosomal-related life activities in cells for purposes other than disease treatment and diagnosis; A3) Prepare products for labeling and / or locating lysosomes in cells; A4) Prepare products for monitoring lysosomal-related life activities in cells.

[0020] Fourthly, the present invention provides an application of the lysosomal fluorescent probe described above in the preparation of products targeting lysosomes.

[0021] Fifthly, the present invention provides the application of the lysosomal fluorescent probe described above in the preparation of products for lysosomal fluorescence imaging.

[0022] According to some embodiments of the present invention, the lysosome is a lysosome in a healthy living cell; or, the lysosome is an acidic or alkalized lysosome in a cell during a physiological or pathological process; or, the lysosome is a lysosome in a fixed cell.

[0023] The healthy living cells described in this invention refer to cells in a normal physiological state.

[0024] The fixed cells described in this invention refer to cells that have been preserved in their original form and structure through chemical or physical methods, while losing their biological activity (i.e., no longer undergoing metabolic, division, or other life activities).

[0025] The cells in the physiological and pathological process refer to a group of cells that deviate from normal cell characteristics due to abnormalities in morphology, structure, or function caused by various pathogenic factors.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The indole long-chain alkylquinoline salt fluorescent probes described in this invention are a novel type of lysosomal-specific fluorescent probe molecules. Compared with existing lysosomal fluorescent probes, the fluorescent probe molecules of this invention do not contain acidic response groups (amino, dimethylamino, morpholino, piperazine, and phenolic hydroxyl groups, etc.). Moreover, the conjugated structure, long-chain alkyl group, and positive charge work together to allow the fluorescent probe molecules to spontaneously aggregate into nanoparticles (without fluorescence) in aqueous solution. They can be endocytosed into lysosomes. The long-chain alkyl group gives the probe molecules strong membrane affinity, allowing the probe to gradually embed itself into the nonpolar lysosomal membrane in molecular form without dissociation, thereby emitting fluorescence. Therefore, the unique feature of the fluorescent probe of this invention is that it does not depend on the acidity of lysosomes and can image the morphology, number and distribution of lysosomes in biological samples with lysosomal alkalinization, including cells in physiological and pathological processes and fixed cells. The fluorescent probe itself does not fluoresce in water / PBS / culture medium (without serum and antibiotics) solution, but emits red fluorescence after binding with lysosomes in cells. At the same time, the fluorescent probe has good cell permeability, good counterstain compatibility and excellent photostability.

[0027] The immobilizable lysosomal fluorescent probe provided by this invention can be used as a fluorescent probe to label the morphology, number and distribution of lysosomes in cells. It can also be used to image and trace lysosomes when they are alkalized. It can provide a simple and intuitive biological detection reagent for lysosome-related physiological and pathological research and clinical diagnosis. It has a wide range of applications and good effects.

[0028] The immobilizable lysosomal fluorescent probe provided by this invention enables the application of acid-independent lysosomal fluorescent probes in imaging cells, particularly in imaging lysosomal alkalinized biological samples, including cells in physiological and pathological processes and immobilized cells, to study the morphology, quantity, and distribution of lysosomes. Attached Figure Description

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings.

[0030] Figure 1 yes( E UV-Vis absorption and fluorescence spectra of 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide in PBS and DMSO; A is the UV-Vis absorption spectrum of the fluorescent probe Ia of the present invention in PBS and DMSO; B is the fluorescence emission spectrum of the fluorescent probe Ia of the present invention under 500 nm light excitation.

[0031] Figure 2 yes( EConfocal fluorescence microscopy images of live HeLa cells before and after fixation following co-staining with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and the dye-modified lysosome-targeting polymer dextran-Alexa Fluor 647 (Dextran-Alexa Fluor 647, molecular weight approximately 10,000, capable of entering lysosomes and immobilization); A is the fluorescence image of the fluorescent probe Ia of the present invention in live cells; B is the fluorescence image (deep red emission) of dextran-Alexa Fluor 647, which can enter lysosomes, in live cells; C is the superimposed image of A and B; D is the bright field image of live cells; E is the fluorescence image of the fluorescent probe Ia of the present invention in fixed cells; F is the fluorescence image of dextran-Alexa Fluor 647 in fixed cells; G is the superimposed image of E and F; H is the bright field image of fixed cells.

[0032] Figure 3 yes( E Confocal fluorescence microscopy images of live HeLa cells before and after fixation following co-staining with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and the commercial lysosomal green fluorescent probe LysoTracker Green; A is the fluorescence image of the fluorescent probe Ia of the present invention in live cells; B is the fluorescence image of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells; C is the overlay of A and B; D is the bright field image of live cells; E is the fluorescence image of the fluorescent probe Ia of the present invention in fixed cells; F is the fluorescence image of the commercial lysosomal green fluorescent probe in fixed cells; G is the overlay of E and F; H is the bright field image of fixed cells.

[0033] Figure 4 yes( E Confocal fluorescence micrographs of live HeLa cells co-stained with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and the commercial lysosomal green fluorescent probe LysoTracker Green, and treated with ammonium chloride solution; A is the fluorescence image of the fluorescent probe Ia of the present invention in live cells; B is the fluorescence image of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells; C is the bright field image of live cells; D is the fluorescence image of the fluorescent probe Ia of the present invention in cells treated with NH4Cl; E is the fluorescence image of the commercial lysosomal green fluorescent probe in cells treated with NH4Cl; F is the bright field image of cells treated with NH4Cl.

[0034] Figure 5 yes( EConfocal fluorescence microscopy images of live HeLa cells co-stained with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and the commercial lysosomal green fluorescent probe LysoTracker Green, and then treated with chloroquine solution; A is the fluorescence image of the fluorescent probe Ia of the present invention in live cells; B is the fluorescence image of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells; C is the bright field image of live cells; E is the fluorescence image of the fluorescent probe Ia of the present invention in chloroquine-treated cells; F is the fluorescence image of the commercial lysosomal green fluorescent probe in chloroquine-treated cells; G is the bright field image of chloroquine-treated cells.

[0035] Figure 6 yes( E Confocal fluorescence microscopy images of live HeLa cells after co-staining with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and commercial lysosomal near-infrared fluorescent probes (LysoBrite NIR) and mitochondrial green fluorescent probes (MitoTracker Green); A is the fluorescence image of the fluorescent probe Ib of this invention; B is the fluorescence image of the commercial lysosomal near-infrared fluorescent probe LysoBrite NIR; C is the fluorescence image of the commercial mitochondrial green fluorescent probe MitoTracker Green; D is a superimposed image of A, B and C.

[0036] Figure 7 yes( E Confocal fluorescence microscopy images of HeLa cells after co-staining and fixing with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and the mitochondrial green fluorescent probe (MitoTracker Green); A is the fluorescence image of the fluorescent probe Ib of this invention; B is the fluorescence image of the commercial mitochondrial green fluorescent probe; C is the superimposed image of A and B; D is the bright field image of the fixed cells.

[0037] Figure 8 yes( E Fluorescence imaging images of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide after incubation and staining at 37°C and 4°C, respectively; A is the fluorescence image of the fluorescent probe Ib of the present invention stained at 37°C, and B is the fluorescence image of the fluorescent probe Ib of the present invention stained at 4°C.

[0038] Figure 9 yes( EConfocal fluorescence microscopy images of various cell types A549, 4T1, and HEK293 co-stained with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide; A is a fluorescence image of A549 cells stained with the fluorescent probe Ib of the present invention; B is a fluorescence image of 4T1 cells stained with the fluorescent probe Ib of the present invention; C is a fluorescence image of HEK293 cells stained with the fluorescent probe Ib of the present invention; E, F, and G are the bright-field images corresponding to A, B, and C, respectively.

[0039] Figure 10 yes( E )-4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide and lysosomal deep red fluorescent probe (LysoTracker Deep Red) are continuous real-time confocal fluorescence imaging images of live HeLa cells after co-staining; A~D are fluorescence imaging images taken by the 1st, 30th, 60th and 100th times of the fluorescent probe Ia of the present invention, respectively; E~H are fluorescence imaging images taken by the 1st, 30th, 60th and 100th times of the commercial lysosomal deep red fluorescent probe.

[0040] Figure 11 yes( E Super-resolution fluorescence microscopy images of HeLa cells stained with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide; A is a fluorescence image of control group cells stained with the fluorescent probe Ia of this invention; B is a fluorescence image of cells treated with NH4Cl; C is a fluorescence image of cells treated with sucrose. Detailed Implementation

[0041] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0042] In this invention, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0043] It should be understood that in various embodiments of the present invention, the sequence number of each process does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0044] The weights of the relevant components mentioned in this invention specification can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to this invention specification is within the scope disclosed in this invention specification. Specifically, the mass described in this invention specification can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0045] In this specification, EX561nm refers to an excitation wavelength of 561nm; EM580~650nm refers to a receiving and transmitting wavelength range of 580nm to 650nm for fluorescence confocal imaging; other expressions similar to EX561nm and EM580~650nm in this invention represent meanings similar to those of EX561nm and EM580~650nm.

[0046] Example 1 This embodiment discloses a ( E The synthesis of 4-(2-(1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide (Ia) involves the following steps: First, 200 µL of 4-methylquinoline and 470 µL of 1-iodohexadecane were dissolved in ethanol and refluxed at 85°C with stirring for three days under an argon atmosphere. Then, an ethanol solution containing 0.218 g of indole-3-carboxaldehyde was added, stirred thoroughly, and a few drops of piperidine were added; the solution gradually turned red. After reflux for two days, excess solvent was evaporated, and the mixture was cooled. The product was purified by column chromatography using a dichloromethane / methanol mixture as eluent, yielding a red powder, which is (…). E )-4-(2-(1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide, yield approximately 17%.

[0047] ( E 1H NMR data for 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide: 1 HNMR (400 MHz, DMSO- d 6), δ (ppm): 12.13 (s, 1H), 9.10 (d, J = 4.0 Hz, 1H), 8.94 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 16.0 Hz, 1H), 8.40 (m, 2H), 8.30 (d, J = 2.8Hz, 1H), 8.22 (m, 1H), 8.16 (t, J= 7.6 Hz, 1H), 8.01 – 7.92 (m, 2H), 7.50 (m,1H), 7.26 (m, 2H), 4.82 (t, J = 7.2 Hz, 2H), 1.89 (m, 2H), 1.34 (m, 2H), 1.17(m, 24H), 0.80 (t, J = 6.8 Hz, 3H). 13 C NMR (400 MHz, DMSO- d 6) δ (ppm): 154.27,146.45, 139.33, 138.32, 137.89, 135.20, 133.45, 128.97, 126.99, 126.24,125.91, 123.63, 122.01, HRMS: calculated 495.37, found 495.37.

[0048] Example 2 This embodiment discloses a ( E The synthesis of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide (Ib) involves the following steps: First, 200 µL of 4-methylquinoline and 470 µL of 1-iodohexadecane were dissolved in ethanol and refluxed at 85°C with stirring for three days under an argon atmosphere. Then, an ethanol solution containing 0.263 g of 5-methoxy-3-formylindole was added, stirred thoroughly, and several drops of piperidine were added; the solution gradually turned red. After reflux for two days, excess solvent was distilled off, and the mixture was cooled. The product was purified by column chromatography using a dichloromethane / methanol mixture as eluent, yielding a dark red powder, which is (…). E )-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide, yield approximately 19%.

[0049] ( E 1H NMR data for 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide: 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 12.10 (s, 1H), 9.12 (d,J = 4.0 Hz, 1H), 8.95 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 16.0 Hz, 1H), 8.48 – 8.38 (m, 3H), 8.16(t, J = 8.0 Hz, 1H), 8.00 – 7.93 (m, 2H), 7.68 (d, 1H), 7.41 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 4.84 (t, J = 8.0 Hz, 2H), 3.89 (s, 3H), 1.89 (m,2H), 1.36-1.16 (m, 26H), 0.81 (t, J = 6.0 Hz, 3H). 13 C NMR (400 MHz, DMSO- d 6) δ(ppm): 155.72, 154.15, 146.03, 139.19, 138.26, 135.07, 132.67, 132.44,128.76, 127.26, 126.95, 126.11, 119.21, HRMS calculated, 525.38; found, 525.38. Test Example 1 HeLa, A549, 4T1, and HEK293 cell cultures: Cancer cells HeLa, A549, 4T1 and normal cells HEK293 were added to T25 cell culture flasks containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin medium and cultured in a saturated humidity incubator at 37°C and 5% CO2, and passaged every 2-3 days.

[0050] Once the cells reach the logarithmic growth phase, transfer them to confocal glass-bottom culture dishes: Wash the confluent T25 cell culture flasks with PBS, then digest them with 1 mL of trypsin for 1-3 minutes (use 0.25% (v / v) trypsin for HeLa, A549, and 4T1, and 0.025% (v / v) trypsin for HEK293). Remove the trypsin, add fresh culture medium, mix thoroughly, and count the cells. Control the cell density by adding more culture medium, aiming for a final cell concentration of 1×10⁻⁶. 5 Then, the cells were seeded into confocal glass-bottom culture dishes and placed in a 5% CO2 saturated humidity incubator for culture until the cells grew to a coverage of about 70% for cell experiments.

[0051] Test Example 2 (E) Absorption and emission spectra of 4-(2-(1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide (Ia) The fluorescent probe Ia prepared in Example 1 was prepared into a 10 µM solution using phosphate-buffered saline (PBS) and dimethyl sulfoxide (DMSO) as solvents. The absorption and fluorescence emission spectra (EX 500 nm) of the prepared solution were then measured using a UV-Vis absorption spectrometer and a fluorescence spectrometer.

[0052] The results are as follows Figure 1 As shown, Figure 1 In diagram A, the UV-Vis absorption spectra of the fluorescent probe Ia of this invention in PBS and DMSO are shown. Figure 1 Image B shows the fluorescence emission spectrum of the fluorescent probe Ia of this invention under 500 nm light excitation. From... Figure 1 As can be seen from Figure A, the absorption of the fluorescent probe Ia is in the range of 400-600 nm. Compared with the absorption in the aqueous solution PBS, the absorption in the organic solvent DMSO shows a red shift. From... Figure 1 As can be seen from Figure B, the fluorescent probe Ia itself hardly emits light in PBS, but exhibits strong fluorescence in the organic solvent DMSO, with a peak position around 600 nm.

[0053] Test Example 3 (E) Lysosomal fluorescence localization of HeLa cells before and after fixation was observed by staining with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia). The HeLa cells in the two groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then the following staining steps were performed: (1) Incubated for 24 h with a complete culture medium solution of lysosome-targeting polymer dextran-Alexa Fluor 647 (which can endocytose live cell lysosomes) modified with 2 µM commercial deep red fluorescent dye, and washed with PBS; (2) Incubated for 30 min with 1 µM of the fluorescent probe Ia solution of the present invention, washed with PBS, and then incubated in DMEM medium for 30 min; (3) One group of cells was fixed with 4 wt% paraformaldehyde solution for 20 min, and washed with PBS. The two groups of cell samples were observed using a confocal fluorescence microscope for multi-channel fluorescence co-localization observation (fluorescent probe Ia of the present invention EX 561 nm, EM 580~650 nm; dextran-Alexa Fluor 647 EX 638 nm, EM 660~750 nm).

[0054] The results are as follows Figure 2 As shown, Figure 2 Image A shows the fluorescence pattern of the fluorescent probe Ia of this invention in living cells. Figure 2 Image B shows the fluorescence of the commercial dextran-Alexa Fluor 647 in living cells. Figure 2 In the diagram, C is a superimposed image of A and B. Figure 2 D in the image represents the bright-field plot of live cells. Figure 2 E in the image represents the fluorescence pattern of the fluorescent probe Ia of this invention in fixed cells. Figure 2 The middle image (F) shows the fluorescence of the commercial dextran-Alexa Fluor 647 in fixed cells. Figure 2 In the diagram, G is a superimposed image of E and F. Figure 2 H in the image represents a bright-field plot of fixed cells. Figure 2 The excellent fluorescence overlap between A and B indicates that the fluorescence of the fluorescent probe Ia in living cells is completely distributed in the lysosomes. Figure 2 The fluorescence overlap between E and F is also very good, indicating that the fluorescence of the fluorescent probe Ia in fixed cells is also completely distributed in the lysosomes. This result proves that ( E )-4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) is independent of lysosomal acidity and is suitable for lysosomal fluorescence imaging of both live and fixed cells.

[0055] Test Example 4 (E) Comparison of fluorescence imaging of lysosomes before and after fixation of HeLa cells stained with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) and commercial lysosomal probes. The HeLa cells in the two groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then the following staining steps were performed: (1) Incubated with 2 µM commercial lysosomal green fluorescent probe (LysoTracker Green) solution for 30 min, and washed with PBS; (2) Incubated with 1 µM of the present invention's fluorescent probe Ia solution for 30 min, washed with PBS, and then incubated in DMEM medium for 30 min; (3) One group of cells was fixed with 4 wt% paraformaldehyde solution for 20 min, washed with PBS, and placed in DMEM medium. The two groups of cell samples were observed using a confocal fluorescence microscope for multi-channel fluorescence observation (the present invention's fluorescent probe Ia EX 561 nm, EM 600~700 nm; the commercial lysosomal green fluorescent probe EX 488 nm, EM 490~530 nm).

[0056] The results are as follows Figure 3 As shown, Figure 3 Image A shows the fluorescence pattern of the fluorescent probe Ia of this invention in living cells. Figure 3 Image B shows the fluorescence of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells. Figure 3 In the diagram, C is a superimposed image of A and B. Figure 3 D in the image represents the bright-field plot of live cells. Figure 3 E in the image represents the fluorescence pattern of the fluorescent probe Ia of this invention in fixed cells. Figure 3 In the middle F image, a fluorescence pattern of a commercial lysosomal green fluorescent probe in fixed cells is shown. Figure 3 In the diagram, G is a superimposed image of E and F. Figure 3 H in the image represents a bright-field plot of fixed cells. Figure 3 The excellent fluorescence overlap between A and B indicates that the fluorescence of the fluorescent probe Ia in living cells is completely distributed in the lysosomes. Figure 3 The fluorescence of E remains bright. Figure 3 The disappearance of fluorescence in F indicates that the fluorescent probe Ia of this invention can be used for lysosomal imaging of fixed cells, while the commercial lysosomal fluorescent probe LysoTracker Green cannot be used for cell fixation. This result further proves ( E 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) is independent of lysosomal acidity and is suitable for lysosomal fluorescence imaging of both live and fixed cells; the commonly used commercial lysosomal fluorescent probe LysoTracker Green cannot be used for fixed cells.

[0057] Test Example 5 (E) Fluorescence imaging observation of HeLa cells before and after NH4Cl treatment with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia). The HeLa cells in the two groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then the following staining steps were performed: (1) incubated with 2 µM commercial lysosomal green fluorescent probe (LysoTracker Green) solution for 30 min, and washed with PBS; (2) incubated with 1 µM of the fluorescent probe Ia of this invention for 30 min, washed with PBS, and then incubated in DMEM medium for 30 min; (3) one group of cells was treated with 10 mM NH4Cl PBS solution for 30 min. The two groups of cell samples were observed using a confocal fluorescence microscope for multi-channel fluorescence observation (fluorescent probe Ia of this invention EX 561 nm, EM 600~700 nm; commercial lysosomal green fluorescent probe EX 488 nm, EM 490~530 nm).

[0058] The results are as follows Figure 4 As shown, Figure 4 Image A shows the fluorescence pattern of the fluorescent probe Ia of this invention in living cells. Figure 4 Image B shows the fluorescence of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells. Figure 4 C in the middle is the bright-field plot of live cells. Figure 4 D is the fluorescence diagram of the fluorescent probe Ia of this invention in cells treated with NH4Cl. Figure 4 Image E shows the fluorescence of a commercial lysosomal green fluorescent probe in cells treated with NH4Cl. Figure 4 In the middle F, the cell is a bright-field plot of cells treated with NH4Cl. Figure 4 The bright fluorescence in samples A and B indicates that both the fluorescent probe Ia of this invention and the commercial lysosomal green fluorescent probe LysoTracker Green can be used for imaging lysosomes in normal live cells. Figure 4 The fluorescence of D remains bright. Figure 4 The disappearance of fluorescence in E indicates that the fluorescent probe Ia of this invention can be used for lysosomal imaging in NH4Cl-treated cells, while the commercial lysosomal green fluorescent probe LysoTrackerGreen cannot be used in NH4Cl-treated cells. This result proves ( E 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) is independent of lysosomal acidity and can be used for lysosomal fluorescence imaging in physiological and pathological processes; while commonly used commercial lysosomal fluorescent probes cannot be used for lysosomal imaging in physiological and pathological processes.

[0059] Test Example 6 (E) Fluorescence imaging observation of HeLa cells before and after chloroquine treatment with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia). The HeLa cells in the two groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then the following staining steps were performed: (1) incubated with 2 µM commercial lysosomal green fluorescent probe (LysoTracker Green) solution for 30 min, and washed with PBS; (2) incubated with 1 µM of the present invention's fluorescent probe Ia solution for 30 min, washed with PBS, and then incubated in DMEM medium for 30 min; (3) one group of cells was treated with 200 µM chloroquine in PBS solution for 30 min. The two groups of cell samples were observed using a confocal fluorescence microscope for multi-channel fluorescence observation (the present invention's fluorescent probe Ia EX 561nm, EM 600~700nm; the commercial lysosomal green fluorescent probe EX 488nm, EM 490~530nm).

[0060] The results are as follows Figure 5 As shown, Figure 5 Image A shows the fluorescence pattern of the fluorescent probe Ia of this invention in living cells. Figure 5 Image B shows the fluorescence of the commercial lysosomal green fluorescent probe LysoTracker Green in live cells. Figure 5 C in the middle is the bright-field plot of live cells. Figure 5 E is the fluorescence image of the fluorescent probe Ia of this invention in chloroquine-treated cells. Figure 5 Image F shows the fluorescence of the commercial lysosomal green fluorescent probe LysoTracker Green in chloroquine-treated cells. Figure 5 G in the image represents a bright-field plot of cells treated with chloroquine. Figure 5 The bright fluorescence in samples A and B indicates that both the fluorescent probe Ia of this invention and the commercial lysosomal green fluorescent probe LysoTracker Green can be used for imaging lysosomes in normal live cells. Figure 5 The fluorescence of E remains bright. Figure 5 The disappearance of fluorescence in F indicates that the fluorescent probe Ia of this invention can be used for lysosomal imaging in chloroquine-treated cells, while the commercial lysosomal green fluorescent probe LysoTracker Green cannot be used in chloroquine-treated cells. This result proves that ( E 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) is independent of lysosomal acidity and can be used for fluorescence imaging of damaged, non-acidic lysosomes during physiological and pathological processes; while commonly used commercial lysosomal fluorescent probes cannot be used for imaging of damaged, non-acidic lysosomes during physiological and pathological processes.

[0061] Test Example 7 (E) lysosomal localization fluorescence imaging of HeLa cells using 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) HeLa cells in the confocal glass-bottom culture dish prepared in Test Example 1 were washed twice with PBS, and then subjected to the following treatments and staining: (1) Incubated with 5 µM commercial mitochondrial green fluorescent probe MitoTracker Green solution for 30 min, followed by washing with PBS; (2) Incubated with 2 µM commercial lysosomal near-infrared fluorescent probe LysoBrite NIR solution for 30 min, followed by washing with PBS; (3) Incubated with 1 µM (E) Incubate the stained cell samples in a solution of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide fluorescent probe (Ib) for 30 min, then wash with PBS. Perform multi-channel fluorescence co-localization observation using a confocal fluorescence microscope (Ib fluorescent probe of this invention: EX 561 nm, EM 600~650 nm; commercial mitochondrial green fluorescent probe: EX 488 nm, EM 490~530 nm; commercial lysosomal fluorescent probe: EX 638 nm, EM 660~750 nm).

[0062] The results are as follows Figure 6 As shown, Figure 6 In diagram A, the fluorescence spectrum of the fluorescent probe Ib of this invention is shown. Figure 6 Image B is the fluorescence spectrum of the commercial lysosomal near-infrared fluorescent probe LysoBrite (NIR). Figure 6 The middle C image shows the fluorescence of the commercial mitochondrial green fluorescent probe MitoTrackerGreen. Figure 6 D is a superimposed diagram of A, B, and C. Figure 6 The excellent overlap between A and B indicates that the fluorescence of the fluorescent probe Ib of this invention is completely distributed in the lysosomes, and it has excellent compatibility with commercial lysosomal near-infrared fluorescent probe LysoBrite NIR and mitochondrial green fluorescent probe MitoTracker Green counterstaining, proving that ( E 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) can be used for lysosomal staining and fluorescence imaging of cells, and also has good compatibility.

[0063] Test Example 8 (E) Fluorescence imaging of fixed HeLa cells using 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide (Ib). The HeLa cells in the confocal glass-bottom culture dish prepared in Test Example 1 were washed twice with PBS, and then the following treatments and staining were performed: (1) Incubated with 5 µM commercial mitochondrial green fluorescent probe MitoTracker Green solution for 30 min, followed by washing with PBS; (2) Incubated with 1 µM (E)Incubate the cells in 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide fluorescent probe solution for 30 min, then wash with PBS; (3) Fix the cells with 4 wt% paraformaldehyde solution for 20 min, then wash with PBS. Observe the stained cell samples using a confocal fluorescence microscope for multi-channel fluorescence imaging (the fluorescent probe of this invention is IbEX 561nm, EM 600~700nm; the commercial mitochondrial green fluorescent probe is EX 488nm, EM 490~530nm).

[0064] The results are as follows Figure 7 As shown, Figure 7 In diagram A, the fluorescence spectrum of the fluorescent probe Ib of this invention is shown. Figure 7 Image B shows the fluorescence pattern of a commercial mitochondrial green fluorescent probe. Figure 7 In the diagram, C is a superimposed image of A and B. Figure 7 D in the image represents a bright-field plot of fixed cells. Figure 7 China A and Figure 7 The fluorescence of probe B is bright and largely non-overlapping, indicating that the fluorescent probe Ib of this invention can be used for lysosomal fluorescence imaging of fixed cells, and has good compatibility with commercial mitochondrial green fluorescent probes, proving ( E 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) can be used for lysosomal fluorescence imaging of fixed cells that have lost lysosomal acidity, and it has good compatibility.

[0065] Test Example 9 (E) Fluorescence imaging of HeLa live cells stained with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) at 37°C and 4°C. The HeLa cells in the two groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then one group was washed with 1 µM PBS. (E) The -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide fluorescent probe solution was incubated at 37°C for 30 min, and another group was incubated with 1 µM (E) The 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexadecylquinoline iodide fluorescent probe solution was incubated at 4°C for 30 min, followed by washing with PBS. The stained cell samples were then observed using a confocal fluorescence microscope (EX 561 nm, EM 600~700 nm).

[0066] The results are as follows Figure 8 As shown, Figure 8 Image A shows the fluorescence pattern of the fluorescent probe Ib of this invention stained at 37°C. Figure 8Image B shows the fluorescence pattern of the fluorescent probe Ib of this invention stained at 4°C. At low temperatures, endocytosis in cells is inhibited, while free diffusion is unaffected by temperature. Figure 8 The fluorescence intensity of A was significantly higher than that of B. This indicates that the fluorescent probe of this invention mainly enters the cell through endocytosis.

[0067] Test Case 10 (E) Lysosomal fluorescence imaging of A549, 4T1, and HEK293 cells stained with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) A549, 4T1, and HEK293 cells in the confocal glass-bottom culture dish prepared in Test Example 1 were washed twice with PBS, and then rinsed with 1 µM PBS. (E) The stained cell samples were incubated in DMEM solution for 30 min with the -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) fluorescent probe, followed by washing with PBS. The stained cell samples were then observed using a confocal fluorescence microscope for fluorescence imaging.

[0068] The results are as follows Figure 9 As shown, Figure 9 Image A is a fluorescence image of A549 cells stained with the fluorescent probe Ib of this invention. Figure 9 Image B is a fluorescence imaging diagram of 4T1 cells stained with the fluorescent probe Ib of this invention. Figure 9 Fluorescence imaging of HEK293 cells stained with the fluorescent probe Ib of this invention (C). Figure 9 E, F, and G are respectively Figure 9 Brightfield diagrams corresponding to A, B, and C. Figure 9 In the diagram, A, B, and C show bright fluorescence, all located inside the cell, indicating that the fluorescent probe Ib of this invention can be used for lysosomal fluorescence imaging of tumor cells A549 and 4T1 and normal cells HEK293, proving ( E 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ib) is suitable for lysosomal staining and fluorescence imaging of various cell types.

[0069] Test Example 11 (E) 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) stained cells fluorescence imaging photostable. The HeLa cells in the confocal glass-bottom culture dish prepared in Test Example 1 were washed twice with PBS, and then the following treatments and staining were performed: (1) Incubated with 2 µM commercial lysosomal deep red fluorescent probe LysoTracker Deep Red solution for 30 min, and washed with PBS; (2) Incubated with 1 µM (E) Incubate the stained cell samples with a 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) fluorescent probe solution for 30 min, then wash with PBS. Perform 100 consecutive fluorescence imaging observations using a confocal fluorescence microscope (Ia fluorescent probe of this invention: EX 561 nm, EM 580~650 nm; commercial lysosomal deep red fluorescent probe: EX 638 nm, EM 660~750 nm).

[0070] The results are as follows Figure 10 As shown, Figure 10 Images A through D are fluorescence images captured by the fluorescent probe Ia of this invention at the 1st, 30th, 60th, and 100th times, respectively. Figure 10 Images E-H are fluorescence images taken at the 1st, 30th, 60th, and 100th times of a commercial lysosomal deep red fluorescent probe. From... Figure 10 As can be seen from A to D, with the increase of the number of shots, the fluorescence intensity not only did not decrease, but also showed a slight increase in fluorescence; from Figure 10 As can be seen from E to H, the fluorescence intensity decreases significantly with the increase in the number of images captured; this indicates that the fluorescence stability of the fluorescent probe Ia of this invention is excellent, and even better than that of commonly used commercial lysosomal probes. This result demonstrates that the fluorescence imaging of the fluorescent probe Ia of this invention has excellent photostability and can be used for long-term tracking observation of lysosomes.

[0071] Test Example 12 (E) Super-resolution fluorescence imaging of cells stained with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide (Ia) The HeLa cells in the three groups of confocal glass-bottom culture dishes prepared in Test Example 1 were washed twice with PBS, and then the following treatments and staining were performed: (1) 1 µM (E) (1) Incubate with 4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide fluorescent probe solution for 30 min, then wash with PBS; (2) Incubate in DMEM medium for 1 h; (3) The second group is treated with 50 mM NH4Cl solution for 30 min, and the third group is incubated with 80 mM sucrose solution for 30 min. The second and third groups are the pathological groups, and the first group is not treated and serves as the control group. The cell samples from the three groups are observed by super-resolution fluorescence microscopy.

[0072] The results are as follows Figure 11 As shown, Figure 11 Image A is a fluorescence imaging diagram of control group cells stained with the fluorescent probe Ia of this invention. Figure 11 Image B is a fluorescence image of cells treated with NH4Cl. Figure 11 In the middle, C represents a fluorescence image of cells treated with sucrose. Figure 11 The fluorescence of A to C is clear and bright, proving that the fluorescent probe Ia of this invention is suitable for super-resolution fluorescence imaging of lysosomes, and is also suitable for super-resolution imaging observation of damaged lysosomes in physiological and pathological processes.

[0073] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A fixable lysosomal fluorescent probe, characterized in that, The immobilizable lysosomal fluorescent probe is a compound having the structure shown in formula (I): Wherein, the R 1 R is any one of hydrogen and C1-C5 n-alkyl groups; 2 It is any one of hydrogen and C1~C3 alkoxy groups; X is any one of halogen atoms, BF4 and ClO4.

2. The immobilizable lysosomal fluorescent probe according to claim 1, characterized in that, The halogen atom is any one of iodine, bromine, and chlorine.

3. The immobilizable lysosomal fluorescent probe according to claim 1, characterized in that, The C1-C5 n-alkyl group is any one of methyl, ethyl, n-propyl, n-butyl, and n-pentyl.

4. The immobilizable lysosomal fluorescent probe according to claim 1, characterized in that, The alkoxy groups of C1 to C3 are any one of methoxy, ethoxy, and propoxy.

5. The immobilizable lysosomal fluorescent probe according to claim 1, characterized in that, The immobilizable lysosomal fluorescent probe is (E) -4-(2-(1H-indole-3-)vinyl)-1-hexadecylquinoline iodide or (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexadecylquinoline iodide.

6. A method for preparing an immobilizable lysosomal fluorescent probe as described in any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: S1. 4-Methylquinoline and the substituted long-chain alkane shown in formula (III) are placed in a solvent and heated under reflux to produce the intermediate shown in formula (IV); S2. The intermediate, the compound shown in formula (V), and the catalyst are mixed and heated under reflux. After the reflux reaction is completed, impurities are removed to obtain the immobilizable lysosomal fluorescent probe. The catalyst includes piperidine. Equations (III), (IV), and (V) are shown below: 。 7. The application of a fixable lysosomal fluorescent probe as described in any one of claims 1 to 5 in any of aspects A1) to A4): A1) Marking and / or locating lysosomes in cells for purposes other than disease treatment and diagnosis; A2) Monitoring of lysosomal-related life activities in cells for purposes other than disease treatment and diagnosis; A3) Prepare products for labeling and / or locating lysosomes in cells; A4) Prepare products for monitoring lysosomal-related life activities in cells.

8. The use of a fixable lysosomal fluorescent probe as described in any one of claims 1 to 5 in the preparation of products targeting lysosomes in cells.

9. The use of a fixable lysosomal fluorescent probe as described in any one of claims 1 to 5 in the preparation of products for lysosomal fluorescence imaging in cells.

10. The application according to claim 9, characterized in that, The lysosome is a lysosome in a healthy living cell; or, the lysosome is an acidic or alkalized lysosome in a cell during a physiological or pathological process; or, the lysosome is a lysosome in a fixed cell.

Images