An excimer ratio fluorescent probe capable of highly selectively imaging lysosomal polarity in cells, preparation method and application thereof

Abstract

The present invention discloses a kind of excimer ratio fluorescent probe, preparation method and application that can be used for high-selectivity lysosomal polarity imaging in cells, and the fluorescent probe is recorded as Lyso-PRG, and only one-step reaction is required to prepare it, and its synthetic raw materials are easy to obtain, simple to operate, and easy to promote. The fluorescent probe is applied to prepare ratio-type lysosomal intracellular polarity change detection reagent or detection element. The fluorescent probe of the present invention pioneered the use of monomer-excimer imaging mechanism for the design of lysosomal polarity imaging probe, and then the obtained fluorescent probe shows extremely low background noise, ultra-high Stocks shift (130nm) and dual-color ratio fluorescence properties; and based on the basis of the fluorescent probe, by introducing quaternary amine positive charge structure, it can be avoided to be combined with lipid membrane and fat droplet, showing excellent lysosomal selectivity, and then in the sensitivity, resolution and accuracy of lysosomal polarity detection, it shows significant advantages, and it has broad application prospects in lysosomal polarity-related pathological research, drug efficacy evaluation and new drug research and development fields.

Description

Technical Field

[0001] The present 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 intracellular organelles with degradation and recycling functions, play a key role in cellular metabolic regulation, signal transduction, and stress responses. Numerous studies have shown that lysosomal dysfunction is closely associated with various pathological processes. In particular, in cancer, lysosomes play a crucial role in tumor cell proliferation, invasion, and drug resistance. Therefore, real-time monitoring of lysosomal status and its microenvironment, particularly changes in polarity, is crucial for understanding tumor development and progression. Polarity is a key physical property of the organelle microenvironment that not only influences the rate and direction of chemical reactions but also plays a crucial role in cellular physiology and pathology. In particular, changes in lysosomal polarity are closely linked to their function, influencing biological processes such as autophagy and digestion. Studies have confirmed that lysosomal polarity in cancer cells is generally lower than that in normal cells, and this change may serve as a potential biomarker for cancer diagnosis. Therefore, the development of fluorescent probes that can accurately track changes in lysosomal polarity is of great scientific and practical significance.

[0003] Currently, most fluorescent probes that can be used for lysosomal imaging are pH-responsive probes. These probes exploit the significant difference between the acidic environment inside lysosomes (pH: ~5-6) and the cellular microenvironment (pH: ~7-8) to track and locate lysosomes. In contrast, fluorescent probes that reflect changes in lysosomal polarity are relatively scarce. This is mainly because polarity probes are often interfered with by the lipid membranes and lipid droplets that exist in large quantities in cells, making it impossible to accurately locate lysosomes. In addition, most lysosomal polarity-responsive fluorescent probes are "always-on" single-channel probes, and therefore exhibit 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 defects of 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 now 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. The fluorescent probe is denoted as Lyso-PRG, and its chemical structure is shown in the following formula (I):

[0007]

[0008] The luminescence mechanism of the excimer ratio fluorescent probe of the present invention (ie, the probe "lighting up" mechanism) is shown in FIG. Figure 10 As shown in the figure, 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 inside the lysosome induces the probe to "collapse" 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 of the present invention for preparing the above-mentioned excimer ratio fluorescent probe is as follows:

[0010]

[0011] The preparation method comprises the following steps: reacting compound (II) and compound (III) 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 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 1-4 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 excimer ratio fluorescent probe of the present invention is used 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.

[0017] Beneficial effects: Compared with the existing technology, 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 ratiometric fluorescence properties; and based on the fluorescent probe, by introducing a quaternary amine positively charged structure, it avoids binding to lipid membranes and lipid droplets, showing excellent lysosomal selectivity. That is, the fluorescent probe is based on the biological properties of the quaternary amine positively charged 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 localization 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 graph 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 Figure 1 is a graph showing the 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 graph showing the fluorescence spectra of Lyso-PRG in PBS solutions containing 70%, 60%, 50%, 40%, 30%, 20%, and 10% 1,4-dioxane; (b) is a graph showing the 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 Figure 1 shows the particle size of the fluorescent probe Lyso-PRG prepared in Example 1 of the present invention in different solvents; (a) shows the particle size in PBS; (b) shows the particle size in DMEM medium; (c) shows the particle size in 1,4-dioxane; and (d) shows 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] Figure 9 This is a diagram illustrating the luminescence mechanism of the fluorescent probe Lyso-PRG of the present invention;

[0028] Figure 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) is shown in Figure 1. (a) shows the cell viability after 24 h of incubation with HepG2 cells; (b) shows the cell viability after 24 h of incubation with L02 cells.

[0029] Figure 11 This is a hemolytic experiment on the biosafety of the fluorescent probe prepared in Example 1 of the present invention;

[0030] Figure 12 Figure 3: Fluorescence signal intensity changes of Lyso-PRG (5 μM) in HelpG2 cells after incubation at 37°C for 0, 5, 10, 20, and 30 min, respectively, as detected by flow cytometry; (a) is the FITC channel; (b) is the PE channel;

[0031] Figure 13Figure 2 shows the fluorescence signal intensity and signal intensity analysis of Lyso-PRG in the FITC and PE channels of a flow cytometer in HepG2 cells in the Ctrl group (no endocytosis inhibition), the Blank group (no probe), and cells pretreated with CZP, nystatin, amiloride, and low temperature at 4°C for 1 hour. (a) shows the fluorescence signal intensity in the FITC channel, and (b) shows the fluorescence signal intensity in the PE channel. (c) shows the signal intensity in the FITC channel of the flow cytometer for each group in (a). (d) shows the signal intensity analysis in the PE channel of the flow cytometer for each group in (a).

[0032] Figure 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] Figure 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] Figure 16 Sucrose-induced 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 10 minutes of sucrose incubation in the experimental group. DETAILED DESCRIPTION

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

[0036] Experimental materials and equipment used in this invention: Synthetic intermediates (II) and (III) were purchased from Jinan Mingke Biotechnology Co., Ltd. Intermediate (II) was CPC1 reported in the literature, and intermediate (III) was Cou-Flu reported in the literature. Chemical reagents were obtained from MacLean Biochemical Co., Ltd. (Shanghai), Sigma-Aldrich (Shanghai, China), and Aladdin Chemical Co., Ltd. (China). Unless otherwise stated, commercially available reagents were used without further purification (all other chemicals were reagent grade). Deionized (DI) water (18.2 MΩ·cm) was used in the experiments, and the purification equipment was Cascada II.I 10 (Pall Filters Co., Ltd.). High-performance liquid chromatography was performed by Shimadzu Corporation, Japan, and the eluents were CH3CN (0.1% trifluoroacetic acid (TFA)) and water (0.1% TFA). Mass spectral data were acquired on a Finnigan LCQ Advantage ion trap mass spectrometer electrospray ionization mass spectrometer (ESI-MS) (Thermo Fisher Scientific). Ultraviolet-visible (UV-vis) absorption spectra were obtained from a UV-visible spectrophotometer (UV-1800, Shimadzu, Japan), and 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, which 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 in an ice bath, then adding EEDQ (0.6 g, 2.4 mmol) and triphenylphosphine (0.8 g, 3 mmol); and raising the temperature to a high temperature. The reaction is carried out under nitrogen protection for 24 hours, and the reaction is monitored by thin-layer chromatography until completion. After completion of the reaction, the solvent is removed under reduced pressure, and the resulting product is 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, as shown in FIG. Figure 4 As shown in Figure 2, the fluorescence emission wavelength and intensity of the fluorescent probe in PBS and 1,4-dioxane were found to be closely related to the polarity of the solvent. Specifically, a broad signal centered at 522 nm was observed in PBS. In the organic solvent 1,4-dioxane, the maximum emission peak red-shifted to 652 nm.

[0046] Optical performance test 2

[0047] In order to quantify the relationship between the fluorescence spectrum 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%, and 10% 1,4-dioxane solutions) were measured 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 A 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 the 40%-70% 1,4-dioxane-PBS solution. The linear regression equation was Y = -0.992X + 21.752, R 2 =0.992.

[0048] Investigation of probe mechanism 1

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

[0050] Investigation of probe mechanism 2

[0051] In order to further verify the formation of aggregated excimer, 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, namely PBS and DMEM culture medium, with average particle sizes of 98.3nm and 143.7nm, respectively. However, in the organic solvents of 1,4-dioxane and DMSO, the particle size was significantly reduced and the PDI value increased. The results show that Lyso-PRG (5mM) can dissolve in organic solvents and form micellar particles in aqueous solution.

[0052] Investigation of probe mechanism 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, such as Figure 8 The average fluorescence lifetime of the 652 nm peak (non-aggregated state) is 2.09 ns, and the average fluorescence lifetime of the 522 nm peak (aggregated state) is 4.22 ns, which further confirms the formation of excimer.

[0054] Therefore, combining the probe mechanism investigation 1 to the probe mechanism investigation 3, combined Figure 9 It can be seen that the luminescence mechanism of the excimer ratio fluorescent probe of the present invention (i.e., the probe "lights up" mechanism) is: 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 (green) in this form. When it binds to lysosomes, the small polar environment within the lysosomes will induce the probe to "disintegrate" from a nanomorphology to a monomer (monomer) form, at which 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 change of the lysosome can be accurately located and monitored.

[0055] Cell level detection 1

[0056] The cytotoxicity of HepG2 and L02 cells after incubation with different concentrations of Lyso-PRG for 24 h was investigated by MTT assay. Figure 10As shown in Figure 3, the survival rate of both cell lines was greater than 85% at Lyso-PRG concentrations between 0 and 10 μM, with no significant inhibitory effect on either cell line. Based on these results, we selected 5 μM Lyso-PRG for subsequent cell studies, which demonstrated 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 Lyso-PRG solutions of different concentrations with red blood cells 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. Figure 11 shown.

[0059] Cell level detection 3

[0060] 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 minutes. At the same time, the group without Lyso-PRG probe was set as the 0-minute group. 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. Figure 12 As the probe incubation time in cells increased, the fluorescence signals in both the FITC and PE channels gradually increased, and the fluorescence signals in the 20-min and 30-min incubation groups were basically the same, indicating that the optimal incubation time for the probe was 20 min. However, to ensure the accuracy of the experiment, the incubation time for the cell experiment was set at 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) inhibits clathrin-mediated invagination, nystatin inhibits caveolin pathway, and amiloride hydrochloride inhibits Na 2+ / H + conversion (macropinocytosis), and inhibition of energy-dependent endocytosis when cultured at 4°C. Figure 13As shown in the figure, the signal intensity in both the FITC and PE channels of HepG2 cells pretreated at 4°C was significantly lower than that of the untreated Ctrl control group and was close to that of the blank group without Lyso-PRG, indicating that at low temperatures, virtually no probe enters the cells. The fluorescence signal intensities of the other inhibition groups were not significantly different from those of the Ctrl group. Therefore, Lyso-PRG enters cells primarily through energy-dependent endocytosis.

[0063] Lysosome imaging investigation 1

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

[0065] Lysosome imaging investigation 2

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

[0067] Lysosome imaging study 3

[0068] Lysosomal storage disease is associated with lysosomal enzyme deficiency and can be induced by high-concentration sucrose solution, which can cause changes in the polarity of lysosomes in cells. In order to observe the changes in polarity during lysosomal storage disease, sucrose (80mM) was added to the HepG2 cell culture medium and incubated in a 37°C incubator for 10 minutes. Then, the DMEM culture medium containing Lyso-PRG (5μM) was 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. Figure 16As shown, the lysosome volume and fluorescence intensity in the experimental group were reduced, indicating that the polarity of the lysosome increased.

[0069] In summary, the excimer ratiometric fluorescent probe of the present invention can image lysosomal polarity with high selectivity in cells, effectively improving lysosomal targeting and greatly reducing background noise. It can be used to prepare ratiometric intralysosomal polarity change detection reagents or detection elements, which are kits or test strips. This fluorescent probe has broad application prospects in the fields of lysosomal polarity-related pathology 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 can achieve the same technical effect, so it is not further 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 an inert gas protection at 0-120°C for 1-24 hours to prepare the Lyso-PRG fluorescent probe. The amount of the condensing agent added can be 1-4 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-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluorophosphate, O-(benzotriazol-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 compound (II), and includes triphenylphosphine, triphenoxyphosphine, 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 named 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) in the presence of a condensing agent and a complex in an organic solvent under the protection of an inert gas at 0°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, wherein: The amount of the condensing agent added is 1-4 times the molar amount of compound (II), which is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

4. The method for preparing an excimer ratio fluorescent probe according to claim 2, wherein: The added amount of the complex is 1-3 times the molar amount of compound (II), which is triphenylphosphine.

5. The method for preparing an excimer ratio fluorescent probe according to claim 2, wherein: The solvent is anhydrous dichloromethane.

6. The method for preparing an excimer ratio fluorescent probe according to claim 2, wherein: The inert gas is nitrogen.

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

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