Polarity- and viscosity-responsive deep red fluorescent probe for real-time diagnosis and treatment of acute alcoholic liver injury

Abstract

The present invention discloses a method for preparing a polarity-viscosity dual-responsive fluorescent probe that can target mitochondria and lipid droplets, and its application in biological imaging, belonging to the field of chemical analysis and detection technology. Due to its rotatable nature and strong chemical stability, the probe shows high sensitivity and selectivity to viscosity and polarity. It has good biocompatibility and can be used to accurately locate mitochondria and lipid droplets through living cell imaging, distinguish normal cells from cancer cells, and accurately detect polarity and viscosity changes in abnormal cells. More importantly, we have successfully used PPBI to perform in vivo imaging of mice with alcoholic acute liver injury, track fluctuations in polarity and viscosity in the liver, and monitor the treatment process of acute liver injury under drug intervention, which provides a strong visualization guarantee for the diagnosis and treatment of alcoholic acute liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a method for preparing a fluorescent probe with dual polarity and viscosity responses that can target mitochondria and lipid droplets, and its application in biological imaging. Background Art

[0002] Intracellular viscosity is crucial for various diffusion-mediated biological processes, including electron transport, metabolic waste transport, and intracellular / intercellular signal transduction [J.Yin, L.Huang, L.Wu, J.Li, TDJames, W.Lin, Smallmolecule based fluorescent chemosensors for imaging the microenvironmentwithin specific cellular regions, Chem.Soc.Rev.2021, 51: 12098. J.A.Robson, M.Kubankova, T.Bond, R.A.Hendley, A.J.P.White, MKKuimova, J.Wilton-Ely, Simultaneous detection of carbon monoxide and viscosity changes in cells, Angew. Chem. Int. Ed. 2020, 59: 21431. CH Wolstenholme, H. Hu, S. Ye, B. E. Funk, D. Jain, CH Hsiung, G. Ning, Y. Liu, X. Li, X. Zhang, Agg Fluor: fluorogenic toolbox enables direct visualization of the multi-step protein aggregation process in live cells, J. Am. Chem. Soc. 2020, 142: 17515.]. In addition to developing and maintaining cellular homeostasis, polarity is also an integral part of the cellular microenvironment. When cells undergo spatial organization and protein composition activities, including differentiation, migration and proliferation, their polarity will change [KN Wang, LY Liu, D. Mao, S. Xu, CPTan, Q. Cao, ZW Mao, B. Liu, A polarity-sensitive ratiometric fluorescence probe forming onitoring changes in lipid droplets and nucleus during ferroptosis, Angew. Chem. Int. Ed. 2021, 60: 15095.].Viscosity and polarity are related to the initiation and advancement of pathophysiological processes such as inflammation, immunosuppression, and cancer [S.Wang, WXRen, JTHou, M.Won, J.An, X.Chen, J.Shu, JJSKim, Fluorescence imaging of pathophysiological microenvironments, Chem.Soc.Rev.2021,50:8887.R.Li, J.Guo, Y.Duan, X.Liu, L.Gui, Y.Xu, X.Kong, Y.Li, H.Chen, Z.Yuan, Monitoring inflammation-cancer progression by cell viscosity, polarity and leucine aminopeptidase using multicolor fluorescent probe, Chem.Eng.J.2022,435:135043.DIDanylchuk, P.-H.Jouard, ASKlymchenko, Targeted solvatochromic fluorescent probes for imaging lipid order in organelles underoxidative and mechanical stress, J.Am.Chem.Soc.2021,143:912.L.He,LHHe,S.Xu,TB Ren,XXZhang,ZJQin,XBZhang,L.Yuan,Engineering of reversible NIR-IIredox-responsive fluorescent probes for imaging of inflammation in vivo,Angew.Chem.Int.Ed.2022,61:e202211409.]. Sufficient clinical symptoms are not noticed until the disease develops to the middle or late stage, which is one of the factors that make the treatment of various clinical diseases difficult.

[0003] Alcoholic liver disease (ALD) caused by chronic or acute alcohol intake is becoming a major threat to human health worldwide. Acute ALD, as the early stage of ALD, can develop into severe liver lesions such as steatohepatitis, cirrhosis, and hepatocellular carcinoma [K. Wang, R. Guo, X.-Y. Chen, Y.-S. Yang, L.-Q. Qiao, M.-L. Wang, Multifunctional lysosome-targetable fluorescent probe for imaging peroxynitrite in acute liver injury model, Chem. Eng. J. 2023, 455: 140491. H. Tao, J. Guo, Y. Ma, Y. Zhao, T. Jin, L. Gu, Y. Dou, J. Liu, H. Hu, X. Xiong, J. Zhang, Luminescence imaging of acute liver injury by biodegradable and biocompatible nanoprobes, ACS Nano 2020,14:11083.K.Wang,C.Liu,H.Zhu,Y.Zhang,M.Su,X.Wang,M.Liu,X.Rong,B.Zhu,Recent advances in small-molecule fluorescent probes for diagnosis of cancer cells / tissues,Coord.Chem.Rev.2023,477:214946.J.Liu,W.Zhang,C.Zhou,M.Li,X.Wang,W.Zhang,Z.Liu,L.Wu,TDJames,P.Li,B.Tang,Precision navigation of hepatic ischemia-reperfusion injury guided by lysosomal viscosity-activatableNIR-II fluorescence, J.Am.Chem.Soc.2022,144:13586.]. To aid in the diagnosis and treatment of acute alcoholic liver injury, it is crucial to explore the mechanisms of acute alcoholic liver injury and to capture changes in polarity and viscosity at the cellular level. In summary, we hope to design fluorescent probes that can accurately capture changes in polarity and viscosity in cells and organisms, and thus help in the diagnosis and treatment of acute alcoholic liver injury. Summary of the Invention

[0004] The present invention aims to provide a method for preparing a fluorescent probe with dual polarity and viscosity responses that can target mitochondria and lipid droplets, and its application in biological imaging.

[0005] The molecular structure of the fluorescent probe in the present invention is as follows:

[0006]

[0007] The synthesis process of the fluorescent probe in the present invention is as follows:

[0008]

[0009] The preparation steps of the probe PPBI are as follows:

[0010] 4-Bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were dissolved in tetrahydrofuran. Aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium were added sequentially, and the mixture was ultrasonically dissolved, vacuumized, and heated under reflux for approximately 14 hours. After completion of the reaction, the reaction solution was cooled to room temperature and dried to obtain a crude product. The crude product was separated and purified by column chromatography, and dried to obtain a yellow solid product, 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde (A1) (yield: 32.5%).

[0011] Iodomethane was added to a round-bottom flask containing 1,1,2-trimethyl-1H-benz[e]indole and acetonitrile and heated under reflux at 45°C for 6 hours. After the reaction was complete, the mixture was cooled to room temperature. A precipitate formed, which was filtered and washed with ether. The precipitate was then dried in a vacuum oven to obtain 1,1,2,3-tetramethyl-1H-benz[e]indol-3-ium iodide (A2) as a pale yellow solid (yield: 95.6%).

[0012] Weigh A1 and A2 into a round-bottom flask containing anhydrous ethanol, sonicate to dissolve, and reflux at 78°C for 9 hours. Monitor the reaction progress by thin-layer chromatography. After completion, cool the reaction mixture to room temperature. A large amount of precipitate will form and be filtered to obtain the desired product. Dry in a vacuum oven to obtain (E)-2-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)-1,1,3-trimethyl-1H-benz[e]indol-3-ium iodide (PPBI) as a dark purple solid (yield: 62.8%).

[0013] The detection mechanism of the fluorescent probe of the present invention is as follows: in a low-viscosity solvent, the carbon-carbon single bond of the fluorescent probe probe PPBI can rotate freely, allowing the probe to return to the ground state from the excited state as a non-radiative transition, resulting in the attenuation of fluorescence emission. In contrast, in a high-viscosity solvent, the ability to rotate between the carbon-carbon single bonds of the probe is suppressed, causing the probe molecule to exist in a planar molecular conformation and expanding the range of the conjugated system composed of the probe PPBI molecules, thereby significantly enhancing the fluorescence intensity and quantum yield of the probe PPBI. In addition, after introducing a triphenylamine derivative into the positively charged semi-cyanine skeleton and connecting it through a C-C bond, the fluorescent molecule PPBI exhibits solvation color change and is highly sensitive to changes in polarity. Based on this, a new type of bifunctional deep red fluorescent probe was designed and synthesized to reflect changes in polarity and viscosity in different solutions and organisms. Among them, triphenylamine, as a typical aggregation-induced luminescence group, has the ability to target lipid droplets, and quaternary ammonium salt derivatives have the ability to target mitochondria.

[0014] Figure 3 Figures 1 and 2 show the UV-visible absorption spectra (A) and fluorescence emission spectra (B, C) of the probe PPBI in different solvents. 370 nm was selected as the excitation wavelength. Compared to the UV-visible absorption spectrum, the corresponding maximum emission peak shifts blue-shifted from 491 nm (1,4-dioxane) to 443 nm (DMSO) with increasing solvent polarity, demonstrating a clear polarity-dependent behavior. Furthermore, at an excitation wavelength of 575 nm, the fluorescence intensity of the probe PPBI in glycerol is much higher than in other solvents.

[0015] Figure 4 The photophysical properties of the prepared probe PPBI in different solvents are shown, including the maximum absorbance λ abs,max , maximum emission λ em,max , fluorescence quantum yield Φ (Rhodamine B as reference). The above results show that the probe PPBI has a large Stokes shift and has a larger fluorescence quantum yield (close to 30%) in glycerol (a high viscosity solvent) compared to other solvents.

[0016] Figure 5 The fluorescence emission spectra of the fluorescent probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex =400nm), the inset shows the color of the solution under 365nm portable UV lamp irradiation, showing obvious solvatochromism.

[0017] Figure 6 The fluorescence emission spectra of the fluorescent probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex=575nm), the inset shows the Tyndall effect observed under red laser pen illumination.

[0018] Figure 7 The fluorescence probe PPBI showed regular changes in the mixed solution of PBS buffer solution (pH=7.4) and glycerol at different ratios. As the glycerol content increased, the fluorescence intensity of PPBI increased significantly.

[0019] Figure 8 The logarithm of the viscosity and the logarithm of the fluorescence intensity (logI 750 ) showed a good linear relationship, and the linear correlation coefficient R was 0.9996.

[0020] Figure 9 This study examined the photostability of the probe PPBI. Under different temperature conditions, the fluorescence intensity of the probe PPBI in pure PBS (0.89 cp) and in a mixture of 95% glycerol and 5% PBS (438.4 cp) remained stable over 60 minutes, demonstrating the probe's excellent stability.

[0021] Figure 10 The effect of pH on the probe PPBI was studied. In the pH range of 4-10, the probe PPBI maintained relatively stable fluorescence intensity in mixed solutions with different viscosity values ​​(0.89, 7.90, 78.9, 246.7 and 438.4 cp), indicating that the probe PPBI was almost unaffected by pH.

[0022] Figure 11 This is a study on the anti-interference ability of the probe PPBI. 2+ ,Cu 2+ ,Fe 2+ ,Fe 3+ ,K + ,Mn 2+ ,Na + ,Zn 2+ ,F - ,Cl - ,S 2- ,ClO - ,CO3 2- ,HSO3 - , Cys, GSH, and H2O2 had no effect on the fluorescence intensity of PPBI, indicating that the probe PPBI has good anti-interference ability.

[0023] Figure 12The figure shows the cytotoxicity of the probe PPBI. It shows that HeLa cells incubated with different concentrations of PPBI (1μM, 2μM, 5μM, 10μM, and 20μM) maintain high cell survival rates. This suggests that the probe PPBI has low cytotoxicity and can be used in vivo.

[0024] Figure 13 The images show the localization of the prepared probe PPBI to mitochondria and lipid droplets. The Pearson coefficients for mitochondria in the green (polarity) and red (viscosity) channels are 0.95 and 0.96, respectively. The Pearson coefficients for lipid droplets in the green (polarity) and red (viscosity) channels are both 0.95. This demonstrates that the probe PPBI has excellent localization properties for both mitochondria and lipid droplets.

[0025] Figure 14 Shown are confocal fluorescence images and relative fluorescence intensity histograms of the PPBI probe in different cell lines (mouse mammary epithelial cells and mouse breast cancer cells). After the addition of PPBI, both the green and red channels of cancer cells were significantly higher than those of normal cells. This demonstrates the significant potential of the PPBI probe in distinguishing normal from cancer cells.

[0026] Figure 15 Shown are confocal fluorescence images and relative fluorescence intensity histograms of the probe PPBI before and after the presence of lipopolysaccharide (LPS) and dexamethasone. LPS invasion can trigger cellular inflammatory responses, while the addition of dexamethasone can lead to organelle dysfunction. Both conditions resulted in significant increases in fluorescence intensity in the green and red channels. This demonstrates that the probe PPBI responds to changes in the intracellular microenvironment induced by LPS and dexamethasone.

[0027] Figure 16 Shown are confocal fluorescence images and relative fluorescence intensity histograms of the PPBI probe in the presence of different concentrations of a GSH peroxidase 4 inhibitor. The GSH peroxidase 4 inhibitor is a ferroptosis inducer, and its addition causes a decrease in the green channel and an increase in the red channel. This demonstrates that the PPBI probe can be used to detect varying degrees of iron depletion in cells stimulated by GSH peroxidase 4 inhibitors.

[0028] Figure 17 Shown are time-dependent fluorescence imaging and relative fluorescence intensity histograms of the PPBI probe in nude mice and tissue sections following continuous high-dose baijiu consumption. Fluorescence intensities in both the green and red channels were significantly increased in nude mice and tissue sections with alcohol-induced acute liver injury. This demonstrates that the probe can image and monitor polarity and viscosity fluctuations in real time in nude mice with acute alcoholic liver injury.

[0029] Figure 18Shown are fluorescence images and relative fluorescence intensity histograms of the probe PPBI in four groups of nude mice: blank control mice, liver injury mice, silymarin-treated mice, and buffer solution control mice, as well as tissue sections and hematoxylin-eosin-stained sections. The fluorescence intensity of the green and red channels in the liver injury model was significantly enhanced compared to that in blank control mice. The addition of silymarin significantly weakened the fluorescence intensity of both channels. There was no significant change in fluorescence intensity before and after buffer solution injection, eliminating the possibility of spontaneous recovery. These results demonstrate the potential application of the probe PPBI in the diagnosis and treatment of acute alcoholic liver injury.

[0030] In summary, using a simple organic synthesis method, we have developed a deep red fluorescent probe that dually responds to polarity and viscosity and targets mitochondria and lipid droplets. The prepared probe, PPBI, exhibits a large Stokes shift, high fluorescence quantum yield, good biocompatibility, and strong anti-interference ability. Cell experiments demonstrated that the prepared probe can precisely localize lipid droplets and mitochondria with low cytotoxicity, distinguish normal cells from cancer cells, and sensitively monitor fluctuations in the abnormal intracellular microenvironment. Furthermore, biological experiments demonstrated that the probe, PPBI, effectively detects changes in polarity and viscosity in a nude mouse model of acute alcoholic liver injury, suggesting important implications for the diagnosis and treatment of acute alcoholic liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation route of probe PPBI.

[0032] Figure 2 Mechanism of polarity and viscosity detection by probe PPBI.

[0033] Figure 3 UV-visible absorption spectra (A) and fluorescence emission spectra (B: λ ex = Normalized fluorescence spectrum C at 370 nm: λ ex = fluorescence spectrum at 575 nm). Probe concentration: 10 μM.

[0034] Figure 4 Photophysical properties of probe PPBI in different solvents (including maximum absorbance λ abs,max , maximum emission λ em,max , fluorescence quantum yield Φ).

[0035] Figure 5 Fluorescence emission spectra of probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex =400 nm), the inset shows the color of the solution under 365 nm hand-held UV lamp irradiation.

[0036] Figure 6 Fluorescence emission spectra of probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex =575nm), the inset shows the Tyndall effect observed under red laser pen illumination.

[0037] Figure 7 Fluorescence emission spectra of probe PPBI in mixed solutions of PBS buffer solution (pH=7.4) and glycerol at different ratios (λ ex =575nm).

[0038] Figure 8 The logarithm of the maximum fluorescence intensity of the probe PPBI (logI 750 ) and the linear relationship between the logarithm of viscosity logη (λ ex =575nm).

[0039] Figure 9 Study on the photostability of probe PPBI in solutions at different temperatures and viscosities (λ ex =575nm).

[0040] Figure 10 The fluorescence intensity changes (λ) of the probe PPBI solution at different pH conditions and different viscosity values ex =575nm).

[0041] Figure 11 Study on the anti-interference property of probe PPBI (1-16 are Ca 2+ ,Cu 2+ ,Fe 2+ ,Fe 3+ ,K + ,Mn 2+ ,Na + ,Zn 2+ ,F - ,Cl - ,S 2- ,ClO - ,CO3 2- ,HSO3 - ,Cys,GSH,H2O2,λ ex =575nm).

[0042] Figure 12 Cytotoxicity of the probe PPBI.

[0043] Figure 13 Confocal imaging of the probe PPBI in HeLa cells with commercial organelle localization dyes for mitochondria and lipid droplets.

[0044] Figure 14Confocal fluorescence imaging and relative fluorescence intensity histogram of the probe PPBI in mouse mammary epithelial cells and mouse breast cancer cells.

[0045] Figure 15 Confocal fluorescence imaging and relative fluorescence intensity histogram of probe PPBI before and after the presence of lipopolysaccharide and dexamethasone.

[0046] Figure 16 Confocal fluorescence imaging and relative fluorescence intensity histogram of the probe PPBI in the presence of different concentrations of GSH peroxidase 4 inhibitor.

[0047] Figure 17 Fluorescence imaging and relative fluorescence intensity histogram of the probe PPBI in the nude mouse model of acute alcoholic liver injury and its tissue sections.

[0048] Figure 18 Fluorescence imaging and relative fluorescence intensity bar graphs of the probe PPBI in four groups of nude mice, including blank control mice, liver injury mice, silymarin-treated mice, and buffer solution control mice, as well as tissue sections and hematoxylin-eosin-stained sections.

[0049] Specific implementation examples

[0050] Example 1: Synthesis of Compound A1

[0051] Dissolve 4-bromotriphenylamine and 5-formyl-2-thiopheneboronic acid in THF. Add potassium carbonate aqueous solution and tetrakis(triphenylphosphine)palladium sequentially. Heat under reflux in a vacuum for 12 hours. Cool to room temperature and spin dry to obtain a crude product, which is then dried on a column to yield A1 (32.5%).

[0052] Example 2: Synthesis of Compound A2

[0053] Iodomethane was added dropwise to a round-bottom flask containing 1,1,2-trimethyl-1H-benz[e]indole and acetonitrile, and the mixture was heated under reflux at 45°C for 6 hours. After cooling to room temperature, the precipitate was filtered, washed with ether, and dried to obtain A2 (95.6%).

[0054] Example 3: Synthesis of probe PPBI

[0055] A1 and A2 were weighed and added to a round-bottom flask containing anhydrous ethanol and refluxed at 78°C for 9 hours. After cooling to room temperature, a large amount of precipitate was precipitated, which was filtered and dried to obtain PPBI (62.8%).

[0056] Example 4: Detection of polarity and viscosity in solution using probe PPBI and its application in bioimaging

[0057] Polarity and viscosity detection in solution: Figure 3 The UV-visible absorption and fluorescence emission spectra of the probe PPBI in different solvents (B:λex = Normalized fluorescence spectrum C at 370 nm: λ ex = Fluorescence spectrum at 575 nm. As shown in Figure B, as solvent polarity increases, the corresponding maximum emission peak blue-shifts from 491 nm (1,4-dioxane) to 443 nm (DMSO), demonstrating a clear polarity-dependent behavior. In Figure C, the fluorescence intensity of the probe PPBI in glycerol is much higher than in other solvents. Figure 4 The photophysical properties of the prepared probe PPBI in different solvents are shown in turn, including the maximum absorbance λ abs,max , maximum emission λ em,max , Stokes shift and fluorescence quantum yield Φ. The above results show that PPBI has a larger Stokes shift and a larger fluorescence quantum yield in glycerol than in other solvents. Figure 5 The fluorescence emission spectra of the fluorescent probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex =400nm), the inset shows the color of the solution under 365nm portable UV lamp irradiation, which shows obvious solvatochromism and can respond to solvents of different polarities. Figure 6 The fluorescence emission spectra of the fluorescent probe PPBI in 1,4-dioxane and 70% 1,4-dioxane + 30% H2O mixed solution (λ ex =575nm), the inset shows the Tyndall effect observed under red laser pen illumination. Figure 7 The fluorescence probe PPBI shows regular changes in mixed solutions of different viscosities. With the increase of glycerol content, the fluorescence intensity of PPBI increases significantly. Figure 8 The logarithm of the viscosity and the logarithm of the fluorescence intensity (logI 750 ), and the linear correlation coefficient R was 0.9996. Figure 9 The photostability of the probe PPBI was studied. Under different temperature conditions, the fluorescence intensity of the probe PPBI in pure PBS (0.89 cp) and in a mixture of 95% glycerol and 5% PBS (438.4 cp) remained unchanged over 60 minutes, demonstrating the probe's good stability. Figure 10 The effect of pH on the probe PPBI was studied. In the pH range of 4-10, the probe PPBI maintained relatively stable fluorescence intensity in mixed solutions with different viscosity values ​​(0.89, 7.90, 78.9, 246.7 and 438.4 cp), indicating that the probe PPBI was almost unaffected by pH. Figure 11 This is a study on the anti-interference ability of the probe PPBI.2+ ,Cu 2+ ,Fe 2+ ,Fe 3+ ,K + ,Mn 2+ ,Na + ,Zn 2+ ,F - ,Cl - ,S 2- ,ClO - ,CO3 2- ,HSO3 - , Cys, GSH, and H2O2 had no effect on the fluorescence intensity of PPBI, indicating that the probe PPBI has good anti-interference ability.

[0058] Polarity and viscosity detection biological applications: Figure 12 The figure shows the cytotoxicity of the probe PPBI. It shows that HeLa cells incubated with different concentrations of PPBI (1μM, 2μM, 5μM, 10μM, and 20μM) maintain high cell survival rates, indicating that the probe PPBI has low cytotoxicity. Figure 13 The images show the localization of the PPBI probe to mitochondria and lipid droplets. The Pearson coefficients for mitochondria in the green (polarity) and red (viscosity) channels of PPBI are 0.95 and 0.96, respectively. The Pearson coefficients for lipid droplets in the green (polarity) and red (viscosity) channels of PPBI are both 0.95. This demonstrates that the PPBI probe has excellent localization properties for both mitochondria and lipid droplets. Figure 14 Shown are confocal fluorescence images and relative fluorescence intensity histograms of the PPBI probe in different cell lines (mouse mammary epithelial cells and mouse breast cancer cells). After the addition of PPBI, both the green and red channels of cancer cells were significantly higher than those of normal cells. This demonstrates the significant potential of the PPBI probe in distinguishing normal from cancer cells.

[0059] Example 5

[0060] First, HeLa cells that were not stimulated by drugs were used as a control experimental group. When the probe PPBI in Example 3 was added to cells pretreated with lipopolysaccharide and dexamethasone, HeLa cells showed stronger green fluorescence and red fluorescence. Confocal fluorescence imaging and relative fluorescence intensity histograms before and after the presence of lipopolysaccharide and dexamethasone showed that the invasion of lipopolysaccharide may cause inflammatory responses in cells, and the addition of dexamethasone may lead to organelle dysfunction, both of which will lead to enhanced fluorescence intensity in the green and red channels. This shows that the probe PPBI can respond to changes in the intracellular microenvironment caused by the intervention of lipopolysaccharide and dexamethasone.

[0061] Example 6

[0062] First, HeLa cells that were not stimulated by the drug were used as a control experimental group. The probe PPBI in Example 3 was added to cells in the presence of different concentrations of GSH peroxidase 4 inhibitors, and confocal fluorescence imaging and relative fluorescence intensity histograms were obtained. GSH peroxidase 4 inhibitors are ferroptosis inducers, and their addition weakened the green channel and enhanced the red channel. This shows that the probe PPBI can be used to detect different degrees of iron removal reactions caused by GSH peroxidase 4 inhibitor stimulation of cells.

[0063] Example 7

[0064] A nude mouse model of acute alcoholic liver injury was established by continuously gavage-feeding mice with high doses of alcohol. Two groups of mice were studied: a control group and a model group. The control group received normal feeding and received a tail vein injection of the probe PPBI prior to imaging. The model group received 3g / kg of 56% alcohol-containing liquor twice daily for three consecutive days and then received an injection of the probe PPBI prior to imaging. Figure 17 Shown are fluorescence imaging and relative fluorescence intensity histograms of the probe PPBI in nude mice and tissue sections following continuous high-dose baijiu consumption. Fluorescence intensities in both the red and green channels were significantly increased in nude mice with alcohol-induced acute liver injury and in their tissue sections, while fluorescence intensities in control nude mice were weaker. This demonstrates that the probe can image and monitor polarity and viscosity fluctuations in nude mice with acute alcoholic liver injury in real time.

[0065] Example 8

[0066] Nude mice were divided into four groups: a blank control group, a liver injury model group, a silymarin-treated (treated) group, and a buffer solution control group. In the silymarin-treated group, mice were administered the drug once daily for three consecutive days. In the buffer solution control group, mice were injected with the buffer solution once daily for three consecutive days. Prior to imaging, the animals were injected with the probe PPBI described in Example 3. Figure 18 Shown are fluorescence images and relative fluorescence intensity histograms of the probe PPBI in four groups of nude mice: blank control mice, liver injury mice, silymarin-treated mice, and buffer solution control mice, as well as tissue sections and hematoxylin-eosin-stained sections. The fluorescence intensity of the green and red channels in the liver injury model was significantly enhanced compared to that in blank control mice. The addition of silymarin significantly weakened the fluorescence intensity of both channels. There was no significant change in fluorescence intensity before and after buffer solution injection, eliminating the possibility of spontaneous recovery. These results demonstrate the potential application of the probe PPBI in the diagnosis and treatment of acute alcoholic liver injury.

[0067] In summary, using a simple organic synthesis method, we have developed a deep red fluorescent probe that dually responds to polarity and viscosity and targets mitochondria and lipid droplets. The prepared probe, PPBI, exhibits a large Stokes shift, high fluorescence quantum yield, good biocompatibility, and strong anti-interference ability. Cellular experiments demonstrated that the prepared probe can precisely localize lipid droplets and mitochondria with low cytotoxicity, distinguish normal cells from cancer cells, and sensitively monitor fluctuations in the abnormal intracellular microenvironment. Furthermore, biological experiments demonstrated that the probe PPBI effectively detects fluctuations in polarity and viscosity in a nude mouse model of acute alcoholic liver injury, suggesting important implications for the diagnosis and treatment of acute alcoholic liver injury.

Claims

1. A fluorescent probe PPBI with dual viscosity and polarity responses that can target mitochondria and lipid droplets, its structural formula is:

2. The preparation method of a fluorescent probe PPBI with dual viscosity and polarity response that can target mitochondria and lipid droplets according to claim 1 is as follows: 4-Bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were dissolved in tetrahydrofuran; an aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium were added in sequence, and the mixture was ultrasonically dissolved and vacuumized, and heated under reflux for 14 hours; after completion of the reaction, the reaction solution was cooled to room temperature and dried to obtain a crude product, which was separated and purified by column chromatography and dried to obtain a yellow solid product A1; Iodomethane was added to a round-bottom flask containing 1,1,2-trimethyl-1H-benz[e]indole and acetonitrile and heated under reflux at 45°C for 6 hours. After the reaction was complete, the mixture was cooled to room temperature to generate a precipitate. This was filtered, washed with ether, and dried in a vacuum oven to obtain a light yellow solid A2. Weigh equal amounts of A1 and A2 into a round-bottom flask containing anhydrous ethanol, sonicate to dissolve, and reflux at 78°C for 9 hours. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, cool the reaction mixture to room temperature. A large amount of precipitate will precipitate, which is filtered to obtain the desired product. Dry in a vacuum drying oven to obtain PPBI, a dark purple solid. The synthesis path is as follows:

Images