A lysosomal-targeting polar fluorescent probe, its preparation method and application

Abstract

This invention discloses a method for preparing a lysosome-targeted polar fluorescent probe and its application in bioimaging, belonging to the field of chemical analysis and detection technology. The structure of the polar fluorescent probe is as follows: The lysosome-targeted polar fluorescent probe of this invention exhibits excellent polar sensing properties and significant aggregation-induced emission characteristics. With increasing solvent polarity, the fluorescence emission of the probe PHPT shows a significant redshift and a marked increase in fluorescence intensity. Within the polarizability range of 0.214–0.353, the maximum emission wavelength of the probe PHPT shows a good linear relationship with the solvent polarizability. The probe also exhibits good photostability. More importantly, the polarity detection properties of the probe PHPT are unaffected by pH and various small biological molecules. The probe PHPT has low cytotoxicity and is suitable for bioimaging. PHPT has the ability to precisely target lysosomes and can be used to detect decreased cell polarity and increased polarity during autophagy, and can be used to monitor physiological activities related to changes in intracellular polarity.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for preparing a lysosomal-targeted polar fluorescent probe and its application in bioimaging. Background Technology

[0002] Changes in the microenvironment of biological systems have a significant impact on various biological processes. Detection of related states is crucial for disease diagnosis and analysis, identification of disease pathological mechanisms, and the research and development of new drug targets. Polarity is an important microenvironmental parameter in biological systems, significantly affecting the rate and direction of chemical reactions. Polarity is essential for establishing and reflecting a large number of complex physiological functions and pathological effects, including activating functional proteins and immune responses, triggering signal transduction and membrane rearrangement, changes in the polarity of protein binding, stimulating cell migration and proliferation, influencing protein-protein interactions and enzyme stability, and membrane permeability. Abnormal changes in polarity have been demonstrated to be associated with various diseases such as inflammation, organ failure, and cancer. Therefore, polarity-sensitive dyes can visualize the distribution, changes, and regulation of polarity in biological systems, promoting research on biochemical reactions and deepening our understanding of important biological processes. Furthermore, polarity-sensitive dyes are also used to distinguish key subcellular microstructures.

[0003] Lysosomes play a crucial role in cellular processes including protein degradation, secretion, plasma membrane repair, and autophagy. Lysosomal polarity influences the interaction activity between enzymes and substrates at the cellular level; the level of lysosomal polarity in cancer cells is lower than in normal cells, and polarity changes during lysosomal autophagy. Therefore, developing new methods that can rapidly and sensitively monitor changes in lysosomal polarity is of great significance for cell biology and related pathological research [J.Yin,L.Huang,L.Wu,J.Li,TDJames,W.Lin,Small molecule based fluorescent chemosensors for imaging the microenvironment within specific cellular regions,Chem.Soc.Rev.2021,51:12098.N.Jiang,J.Fan,F.Xu,X.Peng,H.Mu,J.Wang,X.Xiong,Ratiometric fluorescence imaging of cellular polarity:decrease inmitochondrial polarity in cancer cells,Angew.Chem.Int.Ed.2015,54:2510–2514.M.Li,J.Fan,H.Li,J.Du,S.Long,X.Peng,A ratiometric fluorescence probe for lysosomal polarity,Biomaterials]. 2018, 164:98–105. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a lysosomal-targeting polar fluorescent probe and its application in bioimaging.

[0005] The fluorescent probe of this invention has the following molecular structure:

[0006]

[0007] The fluorescent probe synthesis process in this invention is as follows:

[0008]

[0009] A1: 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde;

[0010] B1: 1-(4-(dimethylamino)-2-hydroxyphenyl)ethane-1-one;

[0011] PHPT: (E)-1-(4-(dimethylamino)-2-hydroxyphenyl)-3-(5-(4-(biphenylamino)phenyl)thiophen-2-yl)prop-2-en-1-one.

[0012] The preparation steps of the probe PHPT are as follows:

[0013] 4-Bromotriphenylamine and 5-aldehyde-2-thiopheneboronic acid were placed in a round-bottom flask and dissolved by sonication with anhydrous tetrahydrofuran. Then, aqueous K₂CO₃ and Pd(PPh₃)₄ were added, the mixture was stirred thoroughly, and then heated under argon protection and refluxed for 18 hours. After cooling to room temperature, the mixture was extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: V). 石油醚 :V 乙酸乙酯 =20:1), yielding the yellow product A1.

[0014] Weigh K₂CO₃ and 4'-amino-2'-hydroxyacetophenone into a round-bottom flask, add 5 mL of anhydrous ethanol and sonicate to dissolve, then add iodomethane and mix thoroughly. Heat the mixture to 55°C and reflux for 36 hours. After the reaction is complete, cool the mixture to room temperature, remove the solvent under reduced pressure, and purify by column chromatography with silica gel (V). 石油醚 :V 乙酸乙酯 =1:1), and after vacuum drying for 24 hours, a white solid product was obtained.

[0015] Compounds A1 and B1 were placed in a round-bottom flask, dissolved by sonication with methanol, and then 60% NaOH solution was added. The mixture was heated under reflux for 8 hours. After cooling the mixture to room temperature, ice water was added, and the solution was adjusted to neutral with dilute hydrochloric acid. An orange precipitate formed, which was filtered to obtain the solid. The solid was then purified by column chromatography (eluent V). 石油醚 :V 乙酸乙酯 =20:1), and the product was dried under vacuum to obtain an orange-red product PHPT.

[0016] The detection mechanism of the fluorescent probe in this invention is as follows:

[0017]

[0018] Under low polarity conditions (low water content), the PHPT probe exhibits weak and short-lived fluorescence due to its weak interaction with the solvent, resulting in a dispersed molecular state. In highly polar solvents (high water content), significant charge separation and excited-state energy dissipation may occur due to solvent interaction. Simultaneously, in unfavorable solvents, molecular aggregation occurs, leading to strong and long-lasting fluorescence. This process generates a large Stokes shift, which may be useful for eliminating potential interference from bioluminescence.

[0019] Figure 3 (A) The UV-Vis absorption spectra of the PHPT probe in different solvents. (B) The fluorescence emission spectra of the PHPT probe in different solvents. (C) The normalized fluorescence spectra (λ) of the PHPT probe in different solvents. ex =455nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 (mol / L). Under different polarities, the absorption spectrum of the PHPT probe changes very little, indicating that the dipole moment of the ground-state probe changes very little with polarity. Using 455 nm as the excitation wavelength to test the fluorescence emission spectrum, it was found that with increasing polarity, the emission wavelength of the PHPT probe redshifts significantly, accompanied by an increase in fluorescence intensity.

[0020] Figure 4 The photophysical properties of the prepared probe PHPT in different solvents were demonstrated, including the maximum absorbance λ. abs,max Maximum emission λ em,max The fluorescence quantum yield Φ was also measured. These results indicate that the fluorescence intensity corresponding to the maximum emission wavelength increased by nearly 27-fold from the low-polarity 1,4-dioxane to the high-polarity DMSO. Using fluorescein in 0.1M NaOH as a reference (Φ = 79%), the differences in the photophysical properties of the probe PHPT in different solvents were investigated. The results showed that the fluorescence quantum yield of the probe in DMSO (dimethyl sulfoxide) (Φ = 1.4%) was much greater than that in 1,4-dioxane (Φ = 0.36%). These phenomena indicate that the dipole moment of the probe undergoes a significant change in the excited state.

[0021] Figure 5 (A) Particle size distribution of probe PHPT in 1,4-dioxane. (B) Particle size distribution of probe PHPT in DMSO. The particle size of the probe in DMSO is significantly larger than that in 1,4-dioxane.

[0022] Figure 6 (A) UV-Vis absorption spectra of probe PHPT in different proportions of water and 1,4-dioxane. (B) Fluorescence emission spectra of probe PHPT in different proportions of water and 1,4-dioxane. (C) Emission color coordinate diagram of probe PHPT. (D) Maximum emission wavelength (λ) of probe PHPT. em,max The relationship between λ and solvent polarizability (Δf) ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5(mol / L). With increasing water content, the absorption spectrum of the PHPT probe changed very little. As the water content in the mixed solvent increased from 0% to 30% (increasing polarity), the maximum emission wavelength of the PHPT probe redshifted from 569 nm to 643 nm, and the fluorescence signal corresponding to the maximum emission wavelength increased. Under 365 nm handheld UV light irradiation, the probe emitted orange light in pure 1,4-dioxane and light red light in a mixed solvent of 1,4-dioxane and 30% water, consistent with the results shown by the emission color coordinates. The significant redshift of the maximum emission wavelength in a highly polar environment may be due to increased charge separation of the PHPT probe and dissipation of excited-state energy. Furthermore, with increasing water content, within the solvent polarizability range of 0.214 to 0.353 (2% H2O–20% H2O), the maximum emission wavelength of the PHPT probe showed a good linear relationship with the solvent polarizability (Δf). Figure 2 .6D, R 2 =0.9899). With increasing water content, the fluorescence of the PHPT probe at 640 nm increased by approximately 48-fold. This is contrary to the typical ICT effect and may be due to the intramolecular torsion restriction caused by probe aggregation in the solvent with increasing water content, a poor solvent.

[0023] Figure 7 (A) Particle size distribution of the PHPT probe in 1,4-dioxane. (B) Particle size distribution of the PHPT probe in a mixed solvent of 1,4-dioxane and 30% water. Illustration: Tyndall effect of the probe in different solvents under green laser illumination. The average particle size of the PHPT probe in the 30% water mixed solvent is significantly larger than that in pure 1,4-dioxane. Simultaneously, a significant Tyndall effect of the PHPT probe in the 30% water mixed solvent can be observed under laser illumination. This further indicates aggregate formation, demonstrating that the PHPT probe possesses excellent aggregation-induced emission performance.

[0024] Figure 8 The probe PHPT is shown in the following spectra: (A) UV-Vis absorption spectrum; (B) fluorescence emission spectrum (λ) in solvents at different pH values. ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 The test system consisted of 1,4-dioxane and 30% PBS buffer solutions at different pH values ​​(mol / L). In a mixed solvent of 1,4-dioxane and 30% water, the absorption spectrum of the PHPT probe remained almost unchanged under different pH conditions. The fluorescence spectrum showed minimal change under both acidic (pH=4) and alkaline (pH=10) conditions. The detection properties of the PHPT probe are almost unaffected by pH, and the probe can detect polarity under physiological pH conditions.

[0025] Figure 9 The change in fluorescence intensity (λ) of the probe PHPT at 640 nm over 1 hour is measured in pure 1,4-dioxane and in a mixed solvent of 1,4-dioxane and 30% water. ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 (mol / L). In two different solvents, under continuous irradiation with 450 nm excitation light, the fluorescence intensity of the PHPT probe at 640 nm showed minimal change over 1 hour. This indicates that the PHPT probe exhibits good photostability in both low-polarity and high-polarity environments, and its fluorescence does not decay over time.

[0026] Figure 10 This study investigates the anti-interference capabilities of the PHPT probe. 1-22 represent Blank and Ca... 2+ Cu 2+ Fe 2+ K + Ag + Al 3+ Zn 2+ ,ClO - H2O2, Glycerol, F - NO3 - HCO3 - S 2- HSO3 - I - SO4 2- ,Br - Cys, GSH, Hcy(λ) ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 The test system was PBS buffer solution with a concentration of mol / L and a pH of 7.4. The fluorescence of the probe was almost identical to that in the PBS buffer solution in the presence of various small biological molecules, indicating that the fluorescence of the PHPT probe was not affected by small biological molecules.

[0027] Figure 11 (A) Cell viability statistics. (Incubation time: 12 hours, PHPT probe concentration: 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) (B) Fluorescence imaging of A549 cells after co-incubation with PHPT probe (10 μM) over time (red channel: λ) ex =488nm, λ em =590–680nm, Green Channel: λ ex =488nm, λ em=500–550 nm, scale bar: 25 μm). After co-incubation with different concentrations of PHPT (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) for 12 hours, the survival rate of A549 cells was above 85%. The results indicate that the PHPT probe has low cytotoxicity and is suitable for bioimaging. A549 cells were co-incubated with the PHPT probe, and then a time series was set. Fluorescence images recorded in both the red and green channels showed that the fluorescence intensity of the PHPT probe in A549 cells remained almost unchanged within 30 min, indicating that the PHPT probe has good photostability within cells.

[0028] Figure 12 The image shows (A) the green, red, and blue channels of the probe PHPT co-stained with a commercial lysosomal dye. (B) The fluorescence distribution of the probe in a specific region (red channel: λ). ex =488nm, λ em =590–680nm, Green Channel: λ ex =488nm, λ em =500–550nm, Blue channel: λ ex =408nm, λ em =450–490 nm, scale bar: 10 μm). The images of the probe in the red and green channels overlap well with the images of commercial lysosomal dyes in the blue channel, with colocalization coefficients of 0.87 and 0.89, respectively, indicating that the probe PHPT has good lysosomal targeting ability.

[0029] Figure 13 The images show (A) real-time confocal imaging of A549 cells stained with PHPT (10 μM) after DMSO (3 μL) induction; and (B) a semi-quantitative statistical analysis of fluorescence intensity at different times within 30 min. To verify the feasibility of the PHPT probe in monitoring intracellular polarity changes, A549 cells stained with PHPT were treated with DMSO (3 μL), and fluorescence images of the cells in the red and green channels were recorded every 10 min. After the addition of DMSO, the fluorescence signal in the red channel was significantly weakened, while the fluorescence in the green channel gradually increased over time. This is similar to the phenomenon observed when the probe's polarity decreases in solution, indicating that the addition of DMSO causes a decrease in cell polarity, and that the PHPT probe can be used to monitor changes in cell polarity.

[0030] Figure 14The images show (A) real-time confocal imaging of A549 cells stained with PHPT (10 μM) after lipopolysaccharide (50 μg / mL) induction within 90 min, and (B) a statistical graph of semi-quantitative fluorescence intensity at different time points within 90 min. We further investigated whether the PHPT probe is suitable for monitoring polarity changes induced by inflammation. After staining A549 cells with the PHPT probe, the cells were further treated with lipopolysaccharide (50 μg / mL), and images of the green and red channels were recorded every 30 min. After LPS treatment, the fluorescence of the green channel showed only a slight change over time, while the fluorescence of the red channel gradually weakened. This indicates that LPS-induced inflammatory cell polarity is reduced, and the PHPT probe can be used to monitor polarity changes in an inflammatory cell model.

[0031] Figure 15 The images show (A) real-time confocal imaging of A549 cells stained with PHPT (10 μM) under starvation conditions, and (B) a semi-quantitative statistical analysis of fluorescence intensity at different times within 2 hours. To further investigate whether the probe can be used to monitor autophagy, we established an autophagy model using starvation induction. A549 cells were stained with PHPT and placed in culture dishes containing only PBS, and the fluorescence changes were observed over the next 2 hours. The red channel fluorescence was initially weak, but then significantly increased over time. The green channel fluorescence intensity showed the opposite trend, exhibiting a significant decrease in fluorescence with prolonged starvation. This indicates that lysosomal polarity gradually increases during starvation-induced autophagy. The results of the cell experiments confirm that the PHPT probe has the ability to accurately locate lysosomes and can be used for real-time monitoring of cell polarity changes under physiological conditions of inflammation and autophagy. These results provide a new strategy for real-time, non-destructive monitoring of cell polarity changes under various physiological conditions. Attached Figure Description

[0032] Figure 1 Preparation route of the probe PHPT.

[0033] Figure 2 The polarity response mechanism of the probe PHPT.

[0034] Figure 3 (A) UV-Vis absorption spectra of the PHPT probe in different solvents. (B) Fluorescence emission spectra of the PHPT probe in different solvents. (C) Normalized fluorescence spectra of the PHPT probe in different solvents. (λ) ex =455nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 mol / L)

[0035] Figure 4 Photophysical properties of the probe PHPT in different solvents.

[0036] Figure 5 (A) Particle size distribution of probe PHPT in 1,4-dioxane. (B) Particle size distribution of probe PHPT in DMSO.

[0037] Figure 6 (A) UV-Vis absorption spectra of probe PHPT in different proportions of water and 1,4-dioxane. (B) Fluorescence emission spectra of probe PHPT in different proportions of water and 1,4-dioxane. (C) Emission color coordinate diagram of probe PHPT. (D) Maximum emission wavelength (λ) of probe PHPT. em The relationship between (λmax) and solvent polarizability (Δf). ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 mol / L)

[0038] Figure 7 (A) Particle size distribution of the PHPT probe in 1,4-dioxane. (B) Particle size distribution of the PHPT probe in a mixed solvent of 1,4-dioxane and 30% water. Inset: Tyndall effect images of the probe in different solvents under green laser illumination.

[0039] Figure 8 (A) UV-Vis absorption spectrum of the PHPT probe in solvents at different pH values; (B) fluorescence emission spectrum. ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 mol / L, the test system was 1,4-dioxane + 30% PBS buffer solution at different pH values)

[0040] Figure 9 The fluorescence intensity of the probe PHPT at 640 nm changed over 1 hour in pure 1,4-dioxane and in a mixed solvent of 1,4-dioxane and 30% water. (λ) ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 mol / L)

[0041] Figure 10 Anti-interference study of probe PHPT. 1-22 represent blank, Ca, and other parameters respectively. 2+ Cu 2+ Fe 2+ K + Ag + Al 3+ Zn 2 + ,ClO - H2O2, glycerol, F -NO3 - HCO3 - S 2- HSO3 - I - SO4 2- ,Br - Cys, GSH, Hcy(λ) ex =450nm, slit width: 10nm, 5nm, probe test concentration is 1×10 -5 mol / L, the test system was PBS buffer solution with pH=7.4.

[0042] Figure 11 (A) Cell viability statistics. (Incubation time: 12 hours, PHPT probe concentration: 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) (B) Fluorescence imaging of A549 cells after co-incubation with PHPT probe (10 μM) over time. (Red channel: λ) ex =488nm, λ em =590–680nm, Green Channel: λ ex =488nm, λ em =500–550 nm, scale bar: 25 μm).

[0043] Figure 12 (A) Green, red, and blue channel images of the PHPT probe co-stained with commercial lysosomal dyes. (B) Fluorescence distribution of the probe in a specific region. (Red channel: λ) ex =488nm, λ em =590–680nm, Green Channel: λ ex =488nm, λ em =500–550nm, Blue channel: λ ex =408nm, λ em =450–490 nm, scale bar: 10 μm).

[0044] Figure 13 (A) Real-time confocal imaging of A549 cells stained with PHPT (10 μM) after DMSO (3 μL) induction. (B) Statistical graph of semi-quantitative analysis of fluorescence intensity at different times within 30 min.

[0045] Figure 14 (A) Real-time confocal imaging of A549 cells stained with PHPT (10 μM) and induced by lipopolysaccharide (50 μg / mL) within 90 min. (B) Statistical graph of semi-quantitative analysis of fluorescence intensity at different times within 90 min.

[0046] Figure 15(A) Real-time confocal imaging of A549 cells stained with PHPT (10 μM) probe under starvation conditions. (B) Statistical graph of semi-quantitative analysis of fluorescence intensity at different times within 2 hours.

[0047] Specific implementation examples

[0048] Example 1: Synthesis of compound A1

[0049] 4-Bromotriphenylamine (2.4 mmol, 778 mg) and 5-aldehyde-2-thiopheneboronic acid (2 mmol, 311 mg) were placed in a 50 mL round-bottom flask and dissolved by sonication in 8 mL of anhydrous tetrahydrofuran. Then, 3.2 mL of 2 mol / L K₂CO₃ aqueous solution and Pd(PPh₃)₄ (116 mg, 0.1 mmol) were added, the mixture was stirred thoroughly, and then heated under reflux for 18 hours under argon protection. After cooling to room temperature, the mixture was extracted with dichloromethane and water. The organic phase was dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: V). 石油醚 :V 乙酸乙酯 =20:1), yielding yellow product A1 (249 mg, yield 35.0%). The structural characterization data of A1 are as follows: 1 H NMR (600MHz, DMSO-d6) δ9.86(s,1H),8.00(d,1H),7.69(d,2H),7.60(d,1H),7.36(t,4H),7.13(t,2H),7.09(d,4H),6.96(d,2H). 13 C NMR (151MHz, DMSO-d6) δ183.6,152.9,148.5,146.4,140.9,139.4,129.7,127.4,125.5,124.9,124.1,123.9,121.7.

[0050] Example 2: Synthesis of Compound B1

[0051] Weigh 5 mmol (691.1 mg) of K₂CO₃ and 5 mmol (755.8 mg) of 4'-amino-2'-hydroxyacetophenone into a round-bottom flask, add 5 mL of anhydrous ethanol and sonicate to dissolve, then add 12.5 mL of iodomethane (CH₃I) and mix thoroughly. Heat the mixture to 55 °C and reflux for 36 hours. After the reaction is complete, cool the mixture to room temperature, remove the solvent under reduced pressure, and purify by column chromatography with silica gel (V). 石油醚 :V 乙酸乙酯 =1:1), after vacuum drying for 24 hours, a white solid product (418.1 mg, yield 46.7%) was obtained. The structural characterization data of B1 are as follows: 1H NMR (600MHz, DMSO-d6) δ12.90(s,1H),7.65(d,1H),6.30(dd,1H),6.02(d,1H),3.01(s,6H),2.46(s,3H). 13 C NMR (151MHz, DMSO-d6) δ201.4,164.5,156.3,133.3,109.9,104.6,97.3,26.0.

[0052] Example 3: Synthesis of probe PPBI

[0053] Compounds A1 (0.44 mmol, 155 mg) and B1 (0.44 mmol, 78.2 mg) were placed in a round-bottom flask, dissolved by sonication in 8 mL of methanol, followed by the addition of 1 mL of 60% NaOH solution. The mixture was heated under reflux for 8 hours. After cooling the mixture to room temperature, ice water was added, and the solution was adjusted to neutral with dilute hydrochloric acid. An orange precipitate formed, which was filtered to obtain the solid. The solid was purified by column chromatography (eluent: V). 石油醚 :V 乙酸乙酯 =20:1), and the product was vacuum dried to obtain the orange-red product PHPT (68 mg, yield 30.0%). The structural characterization data of the probe PPBI are as follows: 1 H NMR(600MHz,DMSO-d6)δ13.83(s,1H),7.96(d,1H),7.87(d,1H),7.63(t,4H ),7.50(dd,3H),7.35(t,5H),7.16–7.06(m,8H),6.99(d,2H),3.05(s,8H). 13 C NMR (151MHz, CDCl3) δ189.4,166.2,155.8,148.2,147.4,147.2,139.0,135.4,133.3,131.1,129 .4,127.2,126.7,124.9,123.5,123.6,123.0,118.8,110.6,104.0,98.1,40.0.HR-MSm / z:calcd for C 33 H 28 N₂O₂S[M+H] + ,517.1905; found,517.1952.

[0054] Example 4: Application of the PHPT probe in bioimaging

[0055] Drug- and starvation-induced changes in cell polarity: In the DMSO-induced cell polarity change experiment, cells were first stained with the probe PHPT (10 μM) for 1 hour, washed three times with PBS, and then DMSO was added to a culture dish. Fluorescence images were recorded every 5 minutes (0 min, 5 min, 10 min, 15 min, 20 min, 30 min). The parameters for acquiring cell images were set as follows: red channel: excitation light source 488 nm, collection range 590–680 nm; green channel: excitation light source 488 nm, collection range 500–550 nm.

[0056] In the lipopolysaccharide (LPS)-induced cell polarity change experiment, cells were first stained with the probe PHPT (10 μM) for 1 hour, washed three times with PBS, and then incubated with LPS (50 μg / mL). Fluorescence images were recorded every 30 min, and the changes in fluorescence images within 90 min were recorded (0 min, 30 min, 60 min, 90 min). The parameters for acquiring cell images were set as follows: red channel: excitation light source 488 nm, collection range 590–680 nm; green channel: excitation light source 488 nm, collection range 500–550 nm.

[0057] In the starvation-induced cell polarity change experiment, cells were first stained with the probe PHPT (10 μM) for 1 hour, washed three times with PBS phosphate buffer, and then placed in a dish without culture medium, with a small amount of PBS phosphate buffer added for testing. Fluorescence images and intensity were recorded after 1 hour and 2 hours of cell starvation. The parameters for cell image acquisition were set as follows: red channel: excitation light source 488 nm, collection range 590–680 nm; green channel: excitation light source 488 nm, collection range 500–550 nm.

[0058] The PHPT probe can be used to monitor decreased cell polarity induced by dimethyl sulfoxide and lipopolysaccharide, and can also be further used to monitor increased polarity during autophagy. With its intramolecular charge transfer and aggregation-inducing effects, the PHPT probe provides an effective tool for studying physiological activities related to intracellular polarity changes, and holds promise for providing new avenues for the diagnosis of lysosomes and diseases related to cell polarity changes.

Claims

1. A lysosomal-targeting polar fluorescent probe, PHPT, has the following structural formula:

2. The method for preparing the lysosomal targeted polar fluorescent probe PHPT according to claim 1, characterized in that... The preparation method involves the following steps: 4-Bromotriphenylamine and 5-aldehyde-2-thiopheneboronic acid were placed in a round-bottom flask and dissolved by sonication with anhydrous tetrahydrofuran. Then, 2 mol / L potassium carbonate aqueous solution and Pd(PPh3)4 were added, and the mixture was stirred until homogeneous. The mixture was then heated under argon protection and refluxed for 18 hours. After cooling to room temperature, the mixture was extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was then purified by column chromatography to obtain a yellow product A1. The structural formula of A1 is [insert structural formula here]. K₂CO₃ and 4'-amino-2'-hydroxyacetophenone were weighed and placed in a round-bottom flask. Anhydrous ethanol was added and dissolved by sonication, followed by the addition of iodomethane (CH₃I) and mixing thoroughly. The mixture was heated under reflux for 36 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Column chromatography was performed using silica gel as the eluent, a mixture of petroleum ether and ethyl acetate in a 1:1 volume ratio. After vacuum drying for 24 hours, a white solid product B1 was obtained. The structural formula of B1 is [insert structural formula here]. Compounds A1 and B1 were placed in a round-bottom flask, dissolved by sonication with methanol, and then NaOH solution was added. The mixture was heated under reflux for 8 hours. After cooling the mixed solution to room temperature, ice water was added, and the solution was adjusted to neutral with dilute hydrochloric acid. An orange precipitate was formed, and the solid was obtained by filtration. The solid was purified by column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent, with a volume ratio of petroleum ether to ethyl acetate of 20:

1. The product was dried under vacuum to obtain the orange-red product PHPT.

Images