Excimer proportional fluorescent probe capable of realizing high-selectivity lysosome polarity imaging in cells as well as preparation method and application of excimer proportional fluorescent probe

Abstract

The invention discloses an excimer proportional fluorescent probe capable of realizing high-selectivity lysosome polarity imaging in cells as well as a preparation method and application thereof, the fluorescent probe is marked as Lyso-PRG and is prepared only through one-step reaction, and the fluorescent probe is easy to obtain synthetic raw materials, simple to operate and easy to popularize. The fluorescent probe is applied to preparation of a reagent or a detection element for detecting polarity change in proportional lysosome. According to the fluorescent probe disclosed by the invention, a monomer-excimmer imaging mechanism is creatively applied to the design of the polar imaging probe for the lysosome, so that the obtained fluorescent probe shows extremely low background noise, ultrahigh Stocks displacement (130nm) and double-color proportional fluorescent properties; on the basis of the fluorescent probe, a quaternary amine positive charge structure is introduced, so that the quaternary amine positive charge structure can be prevented from being combined with a lipid membrane and lipid droplets, excellent lysosome selectivity is shown, and further remarkable advantages in sensitivity, resolution and accuracy of lysosome polarity detection are shown; the method has a wide application prospect in the fields of lysosome polarity-related pathological research, efficacy evaluation and new drug research and development.

Description

Technical Field

[0001] The invention belongs to the technical field of lysosome detection in living cells, and in particular relates to an excimer ratio fluorescent probe capable of highly selectively imaging lysosome polarity in cells, a preparation method and an application thereof. Background Art

[0002] Lysosomes, as important organelles with degradation and recycling functions in cells, play a key role in cell metabolism regulation, signal transduction and stress response. At the same time, a large number of studies have shown that lysosomal dysfunction is closely related to a variety of pathological processes, especially in cancer, where lysosomes play a vital role in tumor cell proliferation, invasion and drug tolerance. Therefore, real-time monitoring of lysosomal status and changes in its microenvironment, especially polarity changes, is crucial for understanding the occurrence and development of tumors. Polarity is a key physical property in the organelle microenvironment, which not only affects the rate and direction of chemical reactions, but also plays an important role in the physiological and pathological processes of cells. In particular, changes in lysosomal polarity are closely related to their functions, affecting biological processes such as autophagy and digestion. Studies have confirmed that the polarity of lysosomes in cancer cells is usually lower than that in normal cells, and this change may become a potential biomarker for cancer diagnosis. Therefore, the development of fluorescent probes that can accurately track changes in lysosomal polarity has important scientific and practical significance.

[0003] Currently, most fluorescent probes that can be used for lysosomal imaging are pH-responsive probes, which are based on the significant difference between the acidic environment inside the lysosome (pH: ~5-6) and the cellular microenvironment (pH: ~7-8), thereby tracking and locating the lysosome. In contrast, fluorescent probes that reflect changes in lysosomal polarity are relatively scarce. This is mainly because polarity probes are often interfered by the presence of a large number of lipid membranes and lipid droplets in cells, making it impossible to accurately locate lysosomes. In addition, most lysosomal polarity-responsive fluorescent probes are "always-on" single-channel probes, so most of them show a high background noise ratio and low sensitivity during imaging.

[0004] Based on this, in order to overcome the current scarcity and insufficiency of lysosomal polarity detection probes, especially the poor targeting and high signal-to-noise ratio of lysosomal polarity probes, a fluorescent probe that can be used for highly selective imaging of lysosomes in living cells is studied. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an excimer ratio fluorescent probe, preparation method and application that can be used for highly selective lysosomal polarity imaging in cells. The fluorescent probe is based on the biological properties of quaternary amine positively charged groups and the luminescence mechanism of the excimer fluorescent probe monomer-excimer, which can effectively improve the targeting of lysosomes and greatly reduce background noise.

[0006] Technical solution: The present invention is a fluorescent probe for the ratio of excimers that can highly selectively image polarity changes in lysosomes in cells, characterized in that the fluorescent probe is denoted as Lyso-PRG, and the chemical structure is shown in the following formula (I):

[0007]

[0008] The luminescence mechanism of the excimer ratio fluorescent probe of the present invention (i.e., the probe "lighting up" mechanism) is shown in FIG. Fig.10 As shown, the overall molecular structure of the probe Lyso-PRG has strong hydrophobicity, so it spontaneously aggregates into an excimer (aggregate) form in the large polar environment of the cytoplasm and exhibits emission in this form (green). When it binds to the lysosome, the small polar environment in the lysosome will induce the probe to "disintegrate" from the nanomorphology to the monomer (monomer) form. At this time, the emission wavelength of the probe spontaneously red-shifts to red emission. By changing the color ratio of the probe inside and outside the lysosome, the dynamic displacement of the lysosome can be tracked in real time and the polarity changes of the lysosome can be accurately located and monitored.

[0009] The method for preparing the above-mentioned excimer ratio fluorescent probe of the present invention has the following preparation route:

[0010]

[0011] The preparation method comprises the following steps: reacting compound (II) and compound (III) in a molar ratio of 1:(1-5) under the action of a condensation agent and a complex in an organic solvent, under the protection of an inert gas and at 0-120° C. for 1-24 hours to obtain the Lyso-PRG fluorescent probe.

[0012] Furthermore, the amount of the condensing agent used in the preparation method of the present invention is 1 to 4 times the molar amount of compound (II), which includes 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, thionyl chloride, Carter's condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, 1-[bis(1H-benzotriazolyl)-methyl]-1,1,3,3-tetramethylammonium hydroxide trifluoroacetic acid, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylamino tetrafluorophosphate or phenyldihydrobenzotriazolyl tetrafluorophosphate.

[0013] Furthermore, the amount of the complex used in the preparation method of the present invention is 1-3 times the molar amount of compound (II), which includes triphenylphosphine, triphenoxyphosphine, tri(p-tolyl)phosphine or tri(p-methoxyphenyl)phosphine.

[0014] Furthermore, the solvent used in the preparation method of the present invention is methanol, ethanol, isopropanol, ether, propylene oxide, N,N-dimethylformamide, dimethyl sulfoxide, chloroform or ethyl acetate.

[0015] Furthermore, the inert gas used in the preparation method of the present invention is argon, nitrogen or helium.

[0016] The invention discloses an application of the excimer ratio fluorescent probe in the preparation of a ratio-type lysosomal polarity change detection reagent or detection element, wherein the detection element is a reagent kit or a test paper.

[0017] Beneficial effects: Compared with the prior art, the significant advantages of the present invention are: the fluorescent probe pioneered the use of monomer-excimer imaging mechanism in the design of lysosomal polarity imaging probes, and the resulting fluorescent probe exhibits extremely low background noise, ultra-high Stocks shift (130nm) and dual-color ratio fluorescence properties; and based on the fluorescent probe, by introducing a quaternary amine positive charge structure, it avoids binding to lipid membranes and lipid droplets, and exhibits excellent lysosomal selectivity. That is, the fluorescent probe is based on the biological properties of the quaternary amine positive charge group and the luminescence mechanism of the excimer fluorescent probe monomer-excimer, which can effectively improve the targeting of lysosomes and greatly reduce background noise, thereby showing excellent properties such as high accuracy and high sensitivity in lysosomal positioning and lysosomal polarity monitoring.

[0018] In addition, this fluorescent probe has high molecular structure safety and stability, and has achieved lysosome polarity contrast imaging in normal cells and tumor cells. It has broad application prospects in lysosome polarity-related pathology research, drug efficacy evaluation and new drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The fluorescent probe Lyso-PRG prepared in Example 1 of the present invention 1 H-NMR spectrum;

[0020] Figure 2 The fluorescent probe Lyso-PRG prepared in Example 1 of the present invention 13 C-NMR spectrum;

[0021] Figure 3 This is the ESI-HRMS image of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention;

[0022] Figure 4 The fluorescence spectra of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention in 1,4-dioxane and PBS solutions respectively;

[0023] Figure 5 The graph is a relationship between the fluorescence spectrum signal intensity ratio of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention and the solution polarity; wherein (a) is a fluorescence spectrum of Lyso-PRG in PBS solutions containing 70%, 60%, 50%, 40%, 30%, 20%, and 10% 1,4-dioxane; (b) is a relationship between the ratio of 1,4-dioxane in the solution and the I of Lyso-PRG. 652 / I 522 Linear analysis of fluorescence signal ratios;

[0024] Figure 6 This is a graph showing the critical micelle concentration of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention;

[0025] Figure 7 The particle size diagrams of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention in different solvents; wherein, (a) is the particle size in PBS; (b) is the particle size in DMEM medium; (c) is the particle size in 1,4-dioxane; (d) is the particle size in DMSO;

[0026] Figure 8 The time-fluorescence decay of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention in 90% 1,4-dioxane aqueous solution and 10% 1,4-dioxane aqueous solution respectively;

[0027] Fig. 9 This is a diagram showing the luminescence mechanism of the fluorescent probe Lyso-PRG of the present invention;

[0028] Fig.10 The cytotoxicity of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention at different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20 μM); wherein, (a) is the cell survival rate after incubation with HepG2 cells for 24 h; (b) is the cell survival rate after incubation with L02 cells for 24 h;

[0029] Fig.11 This is a hemolytic experiment of the biological safety of the fluorescent probe prepared in Example 1 of the present invention;

[0030] Fig.12 The fluorescence signal intensity changes of Lyso-PRG (5 μM) added to HelpG2 cells and incubated at 37°C for 0, 5, 10, 20, and 30 minutes, respectively, detected by flow cytometry; (a) is the FITC channel; (b) is the PE channel;

[0031] Fig.13The fluorescence signal intensity and signal intensity analysis diagram of Lyso-PRG in FITC and PE channels of flow cytometer in Ctrl (no endocytosis inhibition), Blank group (without probe) and HepG2 cells pretreated with CZP, nystatin, amiloride and low temperature 4°C for 1h; wherein, (a) is the fluorescence signal intensity in FITC channel, (b) is the fluorescence signal intensity in PE channel; (c) is the signal intensity diagram of each group in (a) in FITC channel of flow cytometer; (d) is the signal intensity analysis diagram of each group in (a) in PE channel of flow cytometer;

[0032] Fig.14 Confocal fluorescence images of HepG2 cells stained with Lyso-PRG (red, green) and DAPI (first row, blue), DND-22 (second row, blue), ER-Tracker Blue-White DPX (third row, blue), and Cou-Flu (fourth row, blue) for 30 minutes;

[0033] Fig.15 Confocal fluorescence images of HepG2 and L02 cells stained with Lyso-PRG (red, green) and commercial lysosomal probe DND-22 (blue) for 30 minutes respectively;

[0034] Fig.16 Sucrose induces changes in intracellular lysosomal polarity; (a) and (b) are confocal fluorescence images of HepG2 cells stained with Lyso-PRG (5 μM) in the Ctrl group; (c) and (d) are confocal fluorescence images of HepG2 cells stained with Lyso-PRG (5 μM) after sucrose incubation for 10 minutes in the experimental group. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] The experimental materials and equipment used in the present invention: the synthetic intermediate (II) and the intermediate (III) were purchased from Jinan Mingke Biotechnology Co., Ltd., and the intermediate (II) was CPC1 reported in the existing literature, and the intermediate (III) was Cou-Flu reported in the existing literature. Chemical reagents were all from McLean Biochemical Co., Ltd. (Shanghai), Sigma-Aldrich (Shanghai, China), and Aladdin Chemical Co., Ltd. (China). Unless otherwise specified, the commercially available reagents used did not require further purification (all other chemicals were reagent grade). The experimental water was deionized (DI) water (18.2MΩ·cm), and the purification equipment was CascadaⅡ.Ⅰ10 (Pall Filter Co., Ltd.). The high performance liquid chromatography was from Shimadzu Corporation of Japan, and the eluent was CH3CN (0.1% trifluoroacetic acid (TFA)) and water (0.1% TFA). Mass spectrometry data were collected from Finnigan LCQ Advantage ion trap mass spectrometer electrospray ionization mass spectrometer (ESI-MS) instrument (Thermo Fisher Scientific). The UV-visible absorption spectra were obtained from a UV-visible spectrophotometer (UV-1800, Shimadzu, Japan), and the fluorescence spectra were obtained from a fluorescence spectrophotometer (FS5, Edinburgh, UK).

[0037] Example 1

[0038] The preparation route of the excimer ratio fluorescent probe of Example 1 that can highly selectively image polarity changes in lysosomes in cells is as follows:

[0039]

[0040] The preparation method comprises the following steps: dissolving compound (II) (0.5 g, 1.2 mmol) and compound (III) (2.3 g, 4 mmol) in 120 mL of anhydrous dichloromethane; cooling the reaction solution to 0°C under an ice bath, then adding EEDQ (0.6 g, 2.4 mmol) and triphenylphosphine (0.8 g, 3 mmol), and heating the mixture to a high temperature. After reacting for 24 hours under nitrogen protection, the reaction was monitored by thin layer chromatography until the reaction was complete. After the reaction was completed, the solvent was removed under reduced pressure, and the obtained product was purified by preparative chromatography to obtain the target probe. 1 HNMR(600MHz,DMSO-d6)δ8.85(s,1H,Ar- H ),8.50(s,1H,Ar- H ),8.25(d,1H,J=7.7Hz,Ar- H ),8.10(d,1H,J=7.7Hz,Ar- H ),7.66(d,1H,J=8.8Hz,Ar- H ),7.46(d,1H,J=8.8Hz,Ar-H ),7.24(s,1H,C= CH ),6.88-6.84(m,3H,Ar-H),6.74-6.69(m,2H,Ar-H),6.53(s,1H,C= CH ),6.42(s,1H,Ar-H),5.29(s,1H,C= CH ),3.47(t,2H,J=6.0Hz,N CH 2CH2CHO),3.37(t,2H,J=4.0Hz,N CH2 ),3.18(t,2H,J=4.0Hz,N CH2 ),2.97(s,6H, CH3 N CH3 ),2.57(t,2H,J=5.4Hz,CH2 CH 2C=O),2.36(t,2H,J=6.7Hz,C=C CH 2CH2),1.76(t,2H,J=6.7Hz,C=CCH2 CH 2),1.28(s,18H,C( CH 3)3); 13 C NMR(151MHz,DMSO-d6)176.37,171.59,168.30,165.08,162.57,156.81,154.69,154.39,15 3.28,152.88,151.24,148.32,144.73,137.18,135.46,129.51,126.15,124.71,124.24,123 .32,119.79,118.89,116.06,110.84,110.79,109.39,105.37,102.60,98.32,97.76,94.70, 81.60,48.73,47.71,40.39,39.09,32.66,29.47,29.16,27.33,27.10,22.07.;MS(ESI):m / z 989.4159[M+H] + , calculated for C 33 H 34 ClN3O6.

[0041] Structural characterization

[0042] The target probe prepared in Example 1 was subjected to structural characterization, and the results obtained were as follows: Figures 1 to 3 The fluorescent probe is denoted as Lyso-PRG, and its chemical structure is shown in the following formula (I):

[0043]

[0044] Optical performance test 1

[0045] The fluorescent probe Lyso-PRG (5 μM, 1 mL) prepared in Example 1 was dissolved in different solvents (PBS and 1,4-dioxane) and its fluorescence spectrum was detected by fluorescence spectrometer. Figure 4 As shown. It was found that the fluorescence emission wavelength and intensity of the fluorescent probe in PBS and 1,4-dioxane are closely related to the polarity of the solvent, that is, a broad signal centered at 522nm was observed in PBS. In the organic solvent 1,4-dioxane, the maximum emission peak will red-shift to 652nm.

[0046] Optical performance test 2

[0047] In order to quantify the relationship between the fluorescence spectral signal intensity ratio and the solution polarity, the fluorescence spectra of Lyso-PRG (5 μM) in PBS (10 mM) and 1,4-dioxane in different ratios (70%, 60%, 50%, 40%, 30%, 20%, 10% 1,4-dioxane solution) were detected at 25 °C. Figure 5 As shown in Figure 2, as the proportion of 1,4-dioxane in the solvent decreased from 70% to 40%, the ratio of the fluorescence signal decreased by 9.2 times, the fluorescence intensity of the emission peak at 652nm decreased, and the emission peak intensity at 522nm increased. The ratio of the two emission peak intensities I 652 / I 522 Linear regression was performed with the ratio of 1,4-dioxane in the solution. It was found that there was a good linear relationship in 40%-70% 1,4-dioxane-PBS solution. The linear regression equation was Y = -0.992X + 21.752, R 2 =0.992.

[0048] Probe mechanism investigation 1

[0049] By measuring the changes in the maximum absorbance at 520-550nm of Lyso-PRG at different concentrations of 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20μM in rhodamine 6G solution (5μM), Figure 6 The critical micelle concentration (CMC) of Lyso-PRG was calculated to further track the excimer formation process. Lyso-PRGλ can be observed when the probe concentration is higher than 2.49 μM. max It is predicted that starting from this concentration, the morphology of Lyso-PRG changes, that is, aggregation occurs.

[0050] Probe mechanism investigation 2

[0051] In order to further verify the formation of aggregated excimers, the particle size of Lyso-PRG in different solvents was investigated. Figure 7 As shown. An appropriate amount of Lyso-PRG (5mM) stock solution was added to PBS (0.01M, pH7.4), DMEM, 1,4-dioxane and DMSO, respectively. After thorough ultrasonic mixing, the freshly prepared test sample was added to a 1-cm quartz cuvette and detected using a Malvern particle size analyzer. Lyso-PRG can form micellar particles with relatively uniform particle size in aqueous solution, i.e., PBS and DMEM culture medium, with average particle sizes of 98.3nm and 143.7nm, respectively, but in 1,4-dioxane and DMSO organic solvents, the particle size was significantly reduced and the PDI value increased. The results show that Lyso-PRG (5mM) can be dissolved in organic solvents and can form micellar particles in aqueous solution.

[0052] Probe mechanism investigation 3

[0053] When the probe molecule is in the excimer polymerization state, the intramolecular motion will be restricted, which will inhibit its non-radiative decay and facilitate the radiative release of light excitation energy. Therefore, the probe molecule Lyso-PRG was tested by fluorescence lifetime experiment. Figure 8 The average fluorescence lifetime of the 652nm peak (non-aggregated state) is 2.09ns, and the average fluorescence lifetime of the 522nm peak (aggregated state) is 4.22ns, which further confirms the formation of excimer.

[0054] Therefore, combining probe mechanism investigation 1 to probe mechanism investigation 3, combined Fig. 9 It can be seen that the luminescence mechanism of the excimer ratio fluorescent probe of the present invention (i.e., the probe "lighting" mechanism) is: the overall molecular structure of the probe Lyso-PRG has strong hydrophobicity, so it spontaneously aggregates into an excimer (aggregation) form in the large polar environment of the cytoplasm, and exhibits emission in this form (green). After it binds to the lysosome, the small polar environment in the lysosome will induce the probe to "disintegrate" from the nano-morphology to the monomer (monomer) form, at which time the emission wavelength of the probe spontaneously red-shifts to red emission. Through the color ratio change of the probe inside and outside the lysosome, the dynamic displacement of the lysosome can be tracked in real time and the polarity change of the lysosome can be accurately located and monitored.

[0055] Cell level detection 1

[0056] The cytotoxicity of HepG2 cells and L02 cells after incubation with different concentrations of Lyso-PRG for 24 h was investigated by MTT assay. Fig.10As shown. In the range of 0-10μM Lyso-PRG, the survival rate of the two cell lines was greater than 85%, and no obvious inhibitory effect was shown on the two cell lines. Based on the above results, we chose 5μM Lyso-PRG to carry out subsequent cell experiments with higher biosafety.

[0057] Cell level detection 2

[0058] In order to further evaluate the biosafety of Lyso-PRG in vivo detection, a hemolytic experiment was used to examine whether Lyso-PRG would damage the red blood cell membrane and cause hemolysis of red blood cells, so as to avoid the hemolytic hazards that may be caused by the probe during in vivo detection. Therefore, we incubated red blood cells with Lyso-PRG solutions of different concentrations and placed them at room temperature for 12 hours. The positive control group was red blood cells resuspended in water. In the group of red blood cells resuspended in water, the cells absorbed water and swelled, and the supernatant turned red, while Lyso-PRG solutions of different concentrations did not cause obvious hemolysis of red blood cells. The supernatant was taken and the absorbance at OD542 was measured with an enzyme marker, and hemolysis (%) was obtained by calculation. Even when the final concentration of the Lyso-PRG probe was 160 μM, no significant hemolysis occurred. Fig.11 shown.

[0059] Cell level detection 3

[0060] In order to determine the optimal incubation time of the probe, DMEM medium containing Lyso-PRG (5 μM) was added to HelpG2 cells and incubated at 37°C for 5, 10, 20, and 30 min. At the same time, the group without Lyso-PRG probe was set as 0 min. The changes in cell incubation time and fluorescence signal intensity under FITC and PE channels were detected by flow cytometry, as shown in Figure 2. Fig.12 As the probe incubation time in the cell increases, the fluorescence signals under the FITC and PE channels gradually increase, and the fluorescence signals of the 20-min and 30-min incubation groups are basically the same, indicating that the optimal incubation time of the probe is 20 min. However, in order to ensure the accuracy of the experiment, the incubation time of the selected cell experiment was set to 30 min.

[0061] Cell level detection 4

[0062] To further understand how the probe Lyso-PRG enters cells, cells were pre-treated for 1 h with four inhibitors of endocytosis mechanisms: chlorpromazine (CZP) to inhibit clathrin-mediated invagination, nystatin to inhibit caveolin pathway, and amiloride hydrochloride to inhibit Na+-mediated invagination. 2+ / H + conversion (macropinocytosis), and inhibition of energy-dependent endocytosis when cultured at 4°C. Fig.13As shown in the figure, it was observed that the signal intensity of HepG2 cells pretreated by 4°C culture under FITC or PE channels was significantly lower than that of the untreated Ctrl control group, and close to the group without Lyso-PRG, that is, the Blank group, indicating that the cells basically had no probe entering the cells at low temperatures. The fluorescence signal intensity of the other inhibition groups was not significantly different from that of the Ctrl group. Therefore, Lyso-PRG mainly enters the cells through energy-dependent endocytosis.

[0063] Lysosome imaging study 1

[0064] In order to confirm whether Lyso-PRG can selectively label lysosomes, lipid droplet probe Cou-Flu, cell nucleus probe DAPI, lysosome probe DND-22, and endoplasmic reticulum probe ER-Tracker Blue-White DPX were used as control dyes for co-staining experiments. Fig.14 As shown. The PCC of lipid droplet probe Cou-Flu is 0.81, the PCC of endoplasmic reticulum probe ER-TrackerBlue-White DPX is 0.83, and the PCC of cell nucleus probe DAPI is 0.65. Only the PCC of lysosome probe DND-22 and Lyso-PRG is 0.92. This shows that Lyso-PRG can be used as an effective tool for lysosome imaging.

[0065] Lysosome imaging study 2

[0066] To further verify that Lyso-PRG can be used for lysosomal imaging in different cell lines, HepG2 and L02 cells were selected as model cell lines for staining experiments. Fig.15 As shown. The DND-22 commercial lysosome probe was used as the control probe. The PCCs of the red channels of HepG2 and L02 and the commercial lysosome probe were 0.97 and 0.91, respectively, indicating that the Lyso-PRG red channel can be used as a lysosome tracking probe. In addition, the red channel light of the liver cancer cell HepG2 was significantly stronger than that of the normal liver cell L02, indicating that the polarity of the lysosomes of liver cancer cells is less than that of normal cells.

[0067] Lysosome imaging study 3

[0068] Lysosomal storage diseases are associated with lysosomal enzyme deficiencies and can be induced by high-concentration sucrose solutions, which can cause changes in the polarity of intracellular lysosomes. In order to observe changes in polarity during lysosomal storage diseases, sucrose (80 mM) was added to the HepG2 cell culture medium and incubated in a 37°C incubator for 10 minutes. The DMEM culture medium containing Lyso-PRG (5 μM) was then replaced and incubated for another 30 minutes as the experimental group. At the same time, the stained cells that were not incubated with sucrose were used as the Ctrl control group. Fig.16As shown, the volume of lysosomes in the experimental group decreased and the fluorescence intensity decreased, indicating that the polarity of lysosomes increased.

[0069] In summary, the excimer ratio fluorescent probe of the present invention can image lysosomal polarity with high selectivity in cells, can effectively improve the targeting of lysosomes and greatly reduce background noise. It can be used to prepare a ratio-type lysosomal intracellular polarity change detection reagent or detection element, and the detection element is a kit or a test paper. The fluorescent probe can have broad application prospects in the fields of lysosomal polarity-related pathological research, drug efficacy evaluation, and new drug development.

[0070] In addition to the above-mentioned Example 1, the fluorescent probe can be successfully prepared according to the preparation method of the present invention, and the same technical effect can be achieved, so it is not described one by one. That is, compound (II) and compound (III) are reacted in a molar ratio of 1: (1-5) in the presence of a condensing agent and a complex, in an organic solvent, under the protection of an inert gas and at 0-120° C. for 1-24 hours to obtain the Lyso-PRG fluorescent probe. Wherein, the amount of the condensing agent added can be 1-4 times the molar amount of the compound (II), which includes 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, thionyl chloride, Carter condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N,N'-dicyclohexylcarbodiimide, 1-[bis(1H-benzotriazolyl)-methyl]-1,1,3,3-tetramethylammonium hydroxide trifluoroacetic acid, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluorophosphate, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylamino tetrafluorophosphate or phenyldihydrobenzotriazolyl tetrafluorophosphate. The amount of the complex added can be 1-3 times the molar amount of the compound (II), which includes triphenylphosphine, triphenylphosphine oxide, tri(p-tolyl)phosphine or tri(p-methoxyphenyl)phosphine. The solvent may be anhydrous dichloromethane, methanol, ethanol, isopropanol, ether, propylene oxide, N,N-dimethylformamide, dimethyl sulfoxide, chloroform or ethyl acetate. The inert gas may be argon, nitrogen or helium.

Claims

1. A fluorescent probe for the ratio of excimers that can be used for highly selective lysosomal polarity imaging in cells, characterized in that: The fluorescent probe is denoted as Lyso-PRG, and its chemical structure is shown in the following formula (I):

2. A method for preparing the excimer ratio fluorescent probe according to claim 1, characterized in that: The preparation route is as follows: The preparation method comprises the following steps: reacting compound (II) and compound (III) in a molar ratio of 1:(1-5) under the action of a condensation agent and a complex in an organic solvent, under the protection of an inert gas and at 0-120° C. for 1-24 hours to obtain the Lyso-PRG fluorescent probe.

3. The method for preparing an excimer ratio fluorescent probe according to claim 2, characterized in that: The condensing agent is added in an amount of 1 to 4 times the molar amount of compound (II), and includes 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, thionyl chloride, Carter's condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, 1-[bis(1H-benzotriazolyl)-methyl]-1,1,3,3-tetramethylammonium hydroxide trifluoroacetic acid, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluorophosphate, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylamino tetrafluorophosphate or phenyldihydrobenzotriazolyl tetrafluorophosphate.

4. The method for preparing an excimer ratio fluorescent probe according to claim 2, characterized in that: The added amount of the complex is 1-3 times the molar amount of compound (II), which includes triphenylphosphine, triphenoxyphosphine, tri(p-tolyl)phosphine or tri(p-methoxyphenyl)phosphine.

5. The method for preparing an excimer ratio fluorescent probe according to claim 2, characterized in that: The solvent is anhydrous dichloromethane, methanol, ethanol, isopropanol, ether, propylene oxide, N,N-dimethylformamide, dimethyl sulfoxide, chloroform or ethyl acetate.

6. The method for preparing an excimer ratio fluorescent probe according to claim 2, characterized in that: The inert gas is argon, nitrogen or helium.

7. Use of the excimer ratio fluorescent probe according to claim 1 in the preparation of a ratiometric lysosomal polarity change detection reagent or detection element, wherein the detection element is a kit or a test paper.

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