Lysosome targeting viscosity response AIE polymer probe as well as preparation method and application thereof

Abstract

The invention discloses an aggregation-induced emission (AIE) polymer probe with lysosome targeting viscosity response as well as a preparation method and application of the AIE polymer probe. According to the probe, tetraphenyl ethylene (TPE) is used as an AIE luminous skeleton, and two hydrophilic flexible side chains are introduced by utilizing the excellent structural modifiability of the tetraphenyl ethylene (TPE). The tail end of each side chain is modified with pyridine positive ions, and the pyridine positive ions can specifically target a lysosome membrane through electrostatic interaction; the three ether groups embedded in the side chain effectively enhance the water solubility of the molecule. According to the invention, TPE and an amphiphilic flexible side chain are used to construct an AIE polymer, a TPE-derived AIE polymer probe (PTPEB-Py) is designed, and a drug action time-lysosome viscosity-fluorescence lifetime (t-eta-tau) three-dimensional linear relationship is constructed through intracellular and extracellular fluorescence lifetime signals so as to visualize a lysosome viscosity change value. Therefore, quantitative detection and visualization of lysosome viscosity change in the mitochondrial autophagy process in cancer cells are realized.

Description

Technical Field

[0001] This invention belongs to the field of medical polymer materials, specifically relating to the preparation of a viscosity-sensitive aggregation-induced emission (AIE) polymer probe based on tetraphenylethylene (TPE) and its application in detecting changes in lysosomal viscosity in cancer cells. Background Technology

[0002] Lysosomes, as vital organelles responsible for the degradation and recycling of substances within cells, play a crucial role in maintaining normal cellular physiological processes. Abnormal changes in lysosomal viscosity can disrupt normal cellular function and are closely related to the development of various diseases, including malignant tumors, diabetes, and Alzheimer's disease. For example, research by Bhattacharyya et al. (J. Phys. Chem. B, 2015, 119, 2149) showed that the viscosity of lysosomes in normal cells is approximately 30 mPa×s, while it increases to approximately 40 mPa×s in lung cancer cells. Therefore, detecting changes in lysosomal viscosity is of great significance for understanding their physiological functions and for the study, diagnosis, and treatment of related diseases.

[0003] Traditional viscosity measurement tools, including cup, rotation, falling ball, and capillary viscometers, are only suitable for macroscopic fluid measurements in vitro and cannot achieve real-time dynamic detection of viscosity at the micro- and nano-scale within living cells. AIE materials, with their viscosity-dependent luminescence behavior, non-invasive labeling ability, high sensitivity, and high brightness, offer innovative solutions for intracellular viscosity detection. Compared to traditional organic fluorescent probes that are prone to aggregation-induced fluorescence quenching, AIE materials effectively overcome this limitation through intramolecular motion restriction mechanisms. AIE molecules, represented by TPE, not only exhibit significantly enhanced fluorescence in high-viscosity microenvironments but also possess excellent structural modifiability, making them ideal for constructing high-performance viscosity probes. For example, Jiang et al. (Anal. Chem., 2018, 90, 8736) developed a class of small-molecule AIE probes with lysosomal targeting capabilities based on TPE for detecting lysosomal viscosity changes during mitophagy. However, existing AIE-based viscosity probe detection methods mainly rely on fluorescence intensity signals, which are easily affected by non-specific factors such as photobleaching, uneven probe concentration distribution, and excitation light fluctuations. Therefore, although they can sense relative changes in lysosomal viscosity, they are difficult to achieve accurate quantitative analysis.

[0004] Unlike fluorescence intensity, fluorescence lifetime is not limited by fluorophore concentration, thus providing more reliable data for viscosity detection in complex cellular environments. Furthermore, traditional small molecule probes generally suffer from poor water solubility and short residence time in subcellular organelles, while polymer probes, with their tunable molecular structures and unique polymeric effects (such as high absorption efficiency and strong fluorescence properties), show significant potential in improving probe stability and detection sensitivity. Therefore, there is an urgent need to develop a lysosome-targeted viscosity-responsive AIE polymer probe and utilize its response to fluorescence lifetime to achieve quantitative visualization of lysosomal viscosity within cancer cells. Summary of the Invention

[0005] The purpose of this invention is to address the limitations of existing detection methods by providing a lysosomal-targeted viscosity-responsive AIE polymer probe, its preparation method, and its applications. This yields a lysosomal probe possessing AIE properties and lysosomal targeting, exhibiting good biocompatibility within cells, and responding to cell viscosity. This allows for the construction of a drug action time-lysosomal viscosity-fluorescence lifetime signal profile using intracellular and extracellular fluorescence lifetime signals. t - η - τ The intrinsic relationship was used to quantitatively visualize the changes in lysosomal viscosity during mitochondrial autophagy in cancer cells.

[0006] The concept of this invention is to develop a fluorescent probe that combines AIE (autoimmune leukogenesis) properties with lysosomal localization capabilities. This probe uses TPE as the AIE luminescent backbone, leveraging its excellent structural modifiability to introduce two hydrophilic flexible side chains. Each side chain is terminally modified with a pyridine cation, enabling specific targeting of the lysosomal membrane via electrostatic interactions; the three ether groups embedded in the side chains effectively enhance the molecule's water solubility. This invention proposes constructing an AIE polymer using TPE and amphiphilic flexible side chains, designing a TPE-derived AIE polymer probe (PTPEB-Py), and constructing it based on intracellular and extracellular fluorescence lifetime signals. t - η - τ A three-dimensional linear relationship is used to quantitatively visualize changes in lysosomal viscosity.

[0007] The term "lysosome targeting" as used in this specification refers to the ability of the AIE polymer probe molecules to specifically stain lysosomes after entering the cell.

[0008] The "lysosomal viscosity change" mentioned in this specification refers to the change in lysosomal viscosity caused by mitochondrial autophagy induced in cancer cells by the anticancer drug dexamethasone.

[0009] The “viscosity responsiveness” described in this specification refers to the fact that the fluorescence intensity and lifetime of the AIE probe increase with increasing viscosity, meaning that the AIE probe can respond to changes in the viscosity of its environment.

[0010] In this invention, the reactants or organic solvents are all existing products that can be purchased from the market or made in-house.

[0011] The lysosome-targeted viscosity-responsive AIE polymer probe provided by this invention has the following molecular structure:

[0012] The preparation method of the above-mentioned lysosome-targeted viscosity-responsive AIE polymer probe provided by the present invention has the following synthetic route:

[0013] The preparation method specifically includes the following steps: Step 1: TiCl4 was added dropwise to a mixture of 4-bromophenyl(4-hydroxyphenyl) ketone, zinc powder, and anhydrous tetrahydrofuran (THF) under low temperature (-78 °C to 0 °C) and nitrogen atmosphere, wherein the molar ratio of 4-bromophenyl(4-hydroxyphenyl) ketone, zinc powder, and TiCl4 was 1:(2~12):(1~2). The mixture was refluxed and stirred at 60 °C to 70 °C for 12 h to 30 h. After the reaction was completed, the reaction was terminated with Na2CO3 solution. The reaction product was extracted with dichloromethane (DCM) and saturated NaCl solution to remove the solvent from the extracted product and purified to obtain a white solid, which was intermediate product 2.

[0014] Step 2: Intermediate product 2, 1,2-bis(2-bromoethoxy)ethane, K2CO3 and KI were added to an acetone solution, wherein the molar ratio of intermediate product 2, 1,2-bis(2-bromoethoxy)ethane, K2CO3 and KI was 100:(400~600):(400~600):(4~6). The mixture was refluxed and stirred at 55 °C~60 °C for 12 h~24 h. After the reaction was completed, the mixture was purified to obtain a white solid, which was intermediate product 3.

[0015] Step 3: In a nitrogen atmosphere, intermediate 3, 2,1,3-benzothiadiazole-4,7-bis(pinacol borate), tetra(triphenylphosphine)palladium, and an aqueous solution of K2CO3 were sequentially added to toluene. The molar ratio of intermediate 3, 2,1,3-benzothiadiazole-4,7-bis(pinacol borate), and tetra(triphenylphosphine)palladium was 100:(100~120):(3~10). The mixture was refluxed and stirred at 85 °C~95 °C for 24 h~36 h. After the reaction was completed, the mixture was dried with anhydrous magnesium sulfate and then purified to obtain an orange solid as intermediate 4.

[0016] Step 4: Under a nitrogen atmosphere, intermediate product 4 was dissolved in anhydrous pyridine and refluxed at 95 °C to 105 °C for 48 h to 60 h. After the organic solvent was removed by rotary evaporation, the product was washed with acetone, filtered, and the precipitate was collected. After vacuum drying, an orange-red solid was obtained, which is the lysosome-targeted viscosity-responsive AIE polymer probe.

[0017] In the above method, step 1 is further terminated with a 10% (w / w) Na2CO3 solution, wherein the volume ratio of THF to Na2CO3 solution is 10:(2~5).

[0018] In the above method, further, step 2 purification is carried out by column chromatography (ethyl acetate: petroleum ether = 1:2, v / v).

[0019] In the above method, further, in step 3, the concentration of the K2CO3 aqueous solution is 2 mol / L, and its volume is the same as that of the toluene solution.

[0020] In the above method, further, the purification process described in step 3 is to purify the residue obtained from the reaction by column chromatography (dichloromethane:methanol = 50:1, v / v).

[0021] This invention also provides the application of the above-mentioned lysosome-targeted viscosity-responsive AIE polymer probe PTPEB-Py in the preparation of a device for visualizing changes in cell lysosome viscosity before and after drug treatment.

[0022] Preferably, the application is achieved by constructing a drug action time-lysosomal viscosity-fluorescence lifetime (DNT). t - η - τ This invention visualizes lysosomal viscosity changes during mitochondrial autophagy in cancer cells by examining their intrinsic relationship with lysosomal viscosity. The AIE polymer probe PTPEB-Py can visualize these viscosity changes through variations in fluorescence intensity and lifetime. Therefore, the lysosomal-targeted viscosity-responsive AIE polymer probe described in this invention provides a potential research tool for lysosomal physiology studies.

[0023] The present invention also provides a compound, characterized in that it has the following structural formula:

[0024] This invention also provides the application of the above-mentioned compounds as lysosomal targeted viscosity-responsive AIE polymer probes.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. The lysosome-targeted viscosity-responsive AIE polymer probe of the present invention utilizes its AIE properties and water solubility to significantly enhance fluorescence intensity and lifetime as viscosity increases, possessing the potential to detect viscosity changes and can be applied to visualize changes in the viscosity of cancer cell lysosomes.

[0026] 2. The AIE polymer probe of this invention establishes a [missing information - likely a function or mechanism] based on intracellular and extracellular fluorescence lifetime signals. t - η - τ A three-dimensional quantitative relationship diagram is used to visualize the changes in lysosomal viscosity, and this process is not affected by non-viscosity-related factors such as photobleaching, probe concentration, and excitation light intensity.

[0027] 3. The lysosome-targeted viscosity-responsive AIE polymer probe of the present invention is prepared by only four steps compared with traditional commercial lysosome dyes. It has a wide range of raw material sources, low cost, and has great advantages in terms of ease of preparation and economy. Attached Figure Description

[0028] Figure 1 The proton NMR spectrum of intermediate product 2 in Example 1 ( 1 (H NMR) image.

[0029] Figure 2 For intermediate product 3 in Example 1 1 H NMR spectrum.

[0030] Figure 3 For intermediate product 4 in Example 1 1 H NMR spectrum.

[0031] Figure 4 The lysosomal targeted viscosity-responsive AIE polymer probe prepared in Example 1 1 H NMR spectrum.

[0032] Figure 5 The fluorescence spectra of PTPEB-Py at different viscosities and the established linear relationship between fluorescence growth factor and viscosity are shown.

[0033] Figure 6 The fluorescence lifetime of PTPEB-Py at different viscosities and the established linear relationship between fluorescence lifetime and viscosity are shown.

[0034] Figure 7 Cell viability after 4 h of staining with different concentrations of PTPEB-Py.

[0035] Figure 8 Cell survival rates after treatment with different concentrations of dexamethasone for 12 h, 24 h, 36 h and 48 h.

[0036] Figure 9 This study investigated the co-localization of PTPEB-Py with commercially available probes in leukemia cells.

[0037] Figure 10 The graph shows the relationship between the mean fluorescence intensity of cells measured by flow cytometry and the treatment time of dexamethasone.

[0038] Figure 11 The graph shows the linear relationship between the fluorescence lifetime of lysosomes and the treatment time of dexamethasone, as measured by two-photon laser confocal microscopy.

[0039] Figure 12 This is to establish a system based on fluorescence lifetime as a node connecting the inside and outside of the cell. t-η-τ Three-dimensional linear relationship diagram (in the figure) t This refers to the duration of drug action. η This refers to lysosomal viscosity. τ (referring to fluorescence lifetime). Detailed Implementation

[0040] The present invention will be further illustrated below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-described invention, and these improvements and adjustments still fall within the scope of protection of the present invention.

[0041] Example 1 This embodiment prepares a lysosome-targeted viscosity-responsive AIE polymer probe.

[0042] Step 1 Under a nitrogen atmosphere, 5.00 g (18.04 mmol) of 4-bromophenyl(4-hydroxyphenyl) methyl ketone and 11.79 g (180.40 mmol) of dry zinc powder were added to a single-necked flask containing 100 mL of anhydrous THF. The flask was placed in an ice bath at 0 °C and TiCl4 (1.42 mL, 21.65 mmol) was slowly added dropwise. After stirring magnetically for 12 h under reflux (66 °C) in an oil bath, 20 mL of 10% Na2CO3 aqueous solution was slowly added to the flask to quench the reaction. The reaction mixture was extracted three times with dichloromethane. The organic phase after extraction was collected, and the solvent was removed using a rotary evaporator. The crude product was purified by column chromatography (with dichloromethane as the mobile phase) to give a white solid as intermediate 2 (2.83 g, 5.42 mmol; yield: 60%). 1 H NMR (600 MHz, DMSO-) d 6, δ, Figure 1): 9.41 (s, 2H, -OH), 7.34–7.31 (m, 4H, Ar-H), 6.89–6.86 (m, 4H, Ar-H), 6.74–6.70 (m, 4H, Ar-H), 6.54–6.52 (d, 4H, Ar-H).

[0043] Step 2 Intermediate product 2 (2.83 g, 5.42 mmol), 1,2-bis(2-bromoethoxy)ethane (7.48 g, 27.10 mmol), K2CO3 (3.75 g, 27.10 mmol), and KI (44.82 mg, 0.27 mmol) were added to a 100 mL single-necked flask. 30 mL of acetone was added as the reaction solvent. The mixture was refluxed (56 °C) and stirred for 12 h. The acetone was removed by rotary evaporation of the reaction solution. The resulting solid was dissolved in DCM and extracted with saturated NaCl solution. The organic solvent was removed by rotary evaporation again. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:2, v / v) to give a white solid as intermediate product 3 (1.88 g, 2.06 mmol; yield: 38%). 1 H NMR (600 MHz, CDCl3, δ, Figure 2 ): 7.22–7.20 (d, 4H, Ar-H), 6.89–6.86 (m, 8H, Ar-H), 6.66–6.64 (d, 4H, Ar-H), 4.06–4.04 (t , 4H, -CH2-), 3.83–3.78 (d, 8H, -CH2-), 3.72–3.68 (m, 8H, -CH2-), 3.45–3.42 (t, 4H, -CH2-).

[0044] Step 3 Under a nitrogen atmosphere, intermediate 3 (1.88 g, 2.06 mmol), 2,1,3-benzothiadiazole-4,7-bis(pinacol borate) (0.87 g, 2.23 mmol), tetra(triphenylphosphine)palladium (0.09 g, 0.08 mmol), K2CO3 solution (2 M, 8.25 mL), and toluene (8.25 mL) were placed in a 50 mL three-necked flask, refluxed in a 90 °C oil bath, and stirred for 24 h. The mixture was then cooled to room temperature and dried over anhydrous magnesium sulfate. The crude product was purified by column chromatography (dichloromethane:methanol = 50:1, v / v), the organic solvent was removed by rotary evaporation, and the product was washed with methanol. The filter cake was obtained by filtration and vacuum drying to give an orange solid product, which was intermediate 4 (0.95 g; yield: 52%). 1 H NMR (600 MHz, CDCl3, δ, Figure 3): 7.81–7.76 (m, 6H, Ar-H), 7.23–7.21 (d, 4H, Ar-H), 7.07–7.06 (d, 4H, Ar-H), 6.72–6.70 (t, 4H, Ar-H ), 4.07 (s, 4H, -CH2-), 3.83–3.78 (m, 8H, -CH2-), 3.70–3.68 (d, 8H, -CH2-), 3.46–3.43 (t, 4H, -CH2-).

[0045] Step 4 Intermediate product 4 (0.95 g) was dissolved in a two-necked flask containing 30 mL of anhydrous pyridine. The oxygen in the flask was removed by purging with N2 three times. The flask was then transferred to an oil bath and refluxed at 100 °C for 48 h. The organic solvent was removed by rotary evaporation. The flask was then washed with acetone, filtered, and the precipitate was collected. The precipitate was dried under vacuum to obtain an orange-red solid as the target product PTPEB-Py (0.90 g; yield: 80%). M w = 9300 g·mol −1 ). 1 H NMR (600 MHz, DMSO-) d 6, δ, Figure 4 ): 9.01 (s, 4H, Ar-H), 8.55 (s, 2H, Ar-H), 8.09 (s, 4H, Ar-H), 7.88–7.85 (t, 6H, Ar-H), 7.19–7.17 (t, 4 H, Ar-H), 7.03 (s, 4H, Ar-H), 6.80 (s, 4H, Ar-H), 3.97–3.90 (d, 8H, -CH2-), 3.61–3.49 (t, 16H, -CH2-).

[0046] Example 2 This embodiment measures the viscosity response of the PTPEB-Py prepared according to the present invention.

[0047] Test method: PTPEB-Py was dissolved in polyethylene oxide of different viscosities ( M w = 8 million) in an aqueous solution, prepared to a concentration of 0.25 mg × mL -1 The test solution was prepared. The fluorescence intensity of PTPEB-Py solutions at different viscosities was measured using a HORIBA FluoroMax-4 fluorescence spectrometer, and a linear relationship between relative fluorescence intensity and viscosity was plotted. The results are shown in [Figure / Table / Illustration]. Figure 5 .

[0048] Results and Analysis: From Figure 5It can be seen that PTPEB-Py exhibits weak fluorescence at low viscosity (3.72 mPa×s); however, its fluorescence intensity increases significantly with increasing solution viscosity. When the viscosity increases to 315.78 mPa×s, the fluorescence intensity increases approximately 25-fold, indicating that PTPEB-Py possesses typical AIE characteristics. Furthermore, the fluorescence intensity of PTPEB-Py shows a good linear relationship with the microenvironment viscosity, R0... 2 =0.99. This further confirms that the molecule is highly responsive to changes in viscosity.

[0049] Example 3 This embodiment measures the fluorescence lifetime of PTPEB-Py prepared in this invention at different viscosities.

[0050] Test method: PTPEB-Py is dissolved in polyethylene oxide of different viscosities ( M w In an aqueous solution containing 8 million (units unspecified), the probe concentration is 0.25 mg × mL. -1 The fluorescence lifetime of PTPEB-Py solutions at different viscosities was measured using a Fluorolog-3 steady-state-transient near-infrared fluorescence microscopy spectrometer, and the linear relationship between fluorescence lifetime and viscosity was plotted. The results are as follows: Figure 6 As shown.

[0051] Results and Analysis: From Figure 6 It can be seen that the fluorescence lifetime of PTPEB-Py exhibits a significant dependence on environmental viscosity. Under low viscosity conditions (3.72 mPa×s), its fluorescence lifetime is 253 ps; as viscosity increases, the fluorescence lifetime gradually extends, reaching 536 ps at a viscosity of 315.78 mPa×s. The fluorescence lifetime of PTPEB-Py shows a good linear relationship with the viscosity of the environment, indicating that the fluorescence lifetime signal of the AIE polymer probe PTPEB-Py synthesized in this invention can sensitively respond to and quantitatively characterize changes in environmental viscosity.

[0052] Example 4 This embodiment measures the cytotoxicity of PTPEB-Py prepared according to the present invention.

[0053] Test method: First, prepare a cell suspension by suspending leukemia cells in a culture medium and adjusting the cell concentration to 1.0 × 10⁻⁶. 6 Next, PTPEB-Py was dissolved in sterile DMSO solution and then added to the suspended cells to form final concentrations of 5, 10, 15, 20, and 25 μg × mL. -1 The working concentration was determined. Finally, cell viability was measured using the CCK-8 assay to assess the potential toxicity of the AIE polymer probe.

[0054] Results and Analysis: Cytotoxicity Test Results ( Figure 7 This indicates that at a concentration of 25 μg·mL -1 After incubating PTPEB-Py with cells for 4 hours, the cell viability remained above 90%, demonstrating that the probe has good biocompatibility.

[0055] Example 5 This embodiment measures the cytotoxicity of dexamethasone (an anticancer drug).

[0056] Test method: First, leukemia cells were resuspended in culture medium and the density was adjusted to 1.0 × 10⁻⁶. 6 Cell suspensions were prepared using a concentration of cells / mL. Dexamethasone was then dissolved in sterile DMSO solution and added to the cell suspension, establishing a final concentration gradient of 0.25–1.50 μM (0.25, 0.50, 0.75, 1.00, 1.25, 1.50 μM). The treated cells were randomly divided into four groups and cultured at 37 °C in a 5% CO2 incubator for 12, 24, 36, and 48 h, respectively. Finally, the cell viability was assessed using the CCK-8 assay to evaluate the cytotoxicity of dexamethasone at different concentrations and time points.

[0057] Results and Analysis: Figure 8 As shown, although increased concentration slightly reduced cell viability, the effect of dexamethasone treatment time was more pronounced. Specifically, after 48 h of drug treatment, the cell viability (approximately 10%) was significantly lower than that after 24 h (approximately 80%). This data indicates that the cytotoxic effect of dexamethasone significantly increased with time. Through analysis of the above dose-time relationship, the combination of 1.00 µM dexamethasone and a treatment time of 24 h was determined as the relatively optimal stimulation parameter for inducing mitophagy. Under these conditions, leukemia cells exhibited significant lysosomal functional perturbation, with a viability of (80.17 ± 5.56)%, while maintaining cell membrane integrity, providing a reliable experimental window for subsequent real-time detection of lysosomal-related viscosity based on PTPEB-Py.

[0058] Example 6 This embodiment measures the colocalization of the PTPEB-Py prepared in this invention with a commercial probe.

[0059] Test method: First, leukemia cells were taken, suspended in culture medium, and the cell concentration was adjusted to 1.0 × 10⁻⁶. 6Cells / mL. Next, lysosome-specific probes (Lyso-Tracker Green, 50 nM, incubated for 1 h, showing green fluorescence), mitochondrial probes (Mito-Tracker Green, 200 nM, incubated for 30 min, showing green fluorescence), and nuclear staining agent (HOE 33342, 100×, incubated for 30 min, showing blue fluorescence) were used, respectively, with PTPEB-Py (25 μg / mL). -1 Cells (2 mL / sample) were co-stained and labeled for 2 hours. Then, the cells were washed three times by centrifugation with PBS buffer to remove excess dye and culture medium. Finally, imaging was performed using an Olympus FV3000 laser confocal microscope. The excitation wavelength for the red channel was set to 405 nm, and the collection wavelength range was 560–660 nm; the excitation wavelength for the green channel was set to 488 nm, and the collection wavelength range was 500–560 nm; the excitation wavelength for the blue channel was set to 405 nm, and the collection wavelength range was 410–450 nm. These steps effectively stained and imaged the cells to observe the co-staining distribution of PTPEB-Py and commercial dyes within the cells.

[0060] Results and Analysis: Figure 9 As shown, in the dual-channel colocalization experiment, when PTPEB-Py was co-stained with Lyso-Tracker Green, the Pearson colocalization coefficient was as high as 0.81, and the fluorescence signal showed a high degree of consistency in spatial distribution. However, the colocalization coefficients of this probe with Mito-Tracker Green and HOE 33342 were only 0.21 and 0.18, respectively, which were significantly lower than the lysosomal colocalization level. This fully demonstrates that the PTPEB-Py probe has significant lysosomal targeting specificity.

[0061] Example 7 This embodiment tests the intracellular viscosity response of PTPEB-Py prepared in this invention.

[0062] Experiment 1 Test method: First, leukemia cells were prepared at a concentration of 1.0 × 10⁻⁶. 6Cells were resuspended in culture medium at a density of cells / mL, and dexamethasone was added to a final concentration of 1.00 μM (DMSO final concentration <0.5%). 18 mL of cell suspension was evenly divided into 9 groups and incubated at 37 °C in a 5% CO2 incubator for 0–24 hours (time gradient: 0, 3, 6, 9, 12, 15, 18, 21, 24 h). Subsequently, 2 h before the treatment endpoint, each group was stained with the PTPEB-Py probe, stained in the dark, and then washed with PBS buffer to remove excess dye and culture medium. Finally, fluorescence intensity was measured using a BD Celesta flow cytometer to observe changes in intracellular lysosomal viscosity.

[0063] Results and Analysis: Figure 10 As shown, the mean fluorescence intensity of PTPEB-Py labeled cells exhibited a significant time-dependent increase with prolonged dexamethasone treatment time. After 24 h of treatment, the fluorescence growth rate reached 43%. This result indicates that lysosomal viscosity increases with treatment time after drug induction, and the PTPEB-Py probe can effectively monitor this change through fluorescence intensity.

[0064] Experiment 2 Test method: First, the concentration of leukemia cells in the culture medium was adjusted to 1.0 × 10⁻⁶. 6 Cells / mL. Then, 14 mL of cell suspension was divided into 7 groups and dexamethasone was added to achieve a final working concentration of 1.00 μM. The cells were incubated at 37°C, 5% CO2 for 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, respectively. After incubation, PTPEB-Py (final working concentration 25 μg / mL) was added to the cells in each group 2 h before the treatment endpoint. -1 Staining was performed in the dark, followed by washing away excess dye and culture medium with PBS buffer. Finally, N-SIM / A1R MP was used. + Fluorescence lifetime was tested using a high-resolution two-photon laser confocal microscope.

[0065] Results and Analysis: Figure 11 As shown, with the extension of dexamethasone treatment time, the fluorescence lifetime of intracellular PTPEB-Py showed a linear increase, from approximately 410 ps before treatment to 599 ps after 24 h. Based on previously established fluorescence lifetime (… τ ) and viscosity ( η The linear relationship curve of lysosomes indicates that the viscosity increased from approximately 73 mPa×s to 680 mPa×s. This was achieved by correlating the intracellular... t - τ Relationships established extracellularly η - τ In relation to this invention, a method for quantitatively visualizing lysosomal viscosity has been successfully constructed. t-η-τ Three-dimensional linear quantization model ( Figure 12 The model clearly shows that as the duration of drug action increases, lysosomal viscosity and fluorescence lifetime increase synchronously, and the two remain linearly correlated. This result verifies that PTPEB-Py can achieve quantitative detection and visualization of lysosomal viscosity through changes in its fluorescence lifetime.

Claims

1. A lysosome-targeted viscosity-responsive AIE polymer probe, characterized in that, Its molecular structure is as follows: 。 2. The method for preparing the lysosome-targeted viscosity-responsive AIE polymer probe according to claim 1, characterized in that, Includes the following steps: Step 1: TiCl4 was added dropwise to a mixture of 4-bromophenyl(4-hydroxyphenyl) ketone, zinc powder, and anhydrous tetrahydrofuran under nitrogen atmosphere at -78 °C to 0 °C, wherein the molar ratio of 4-bromophenyl(4-hydroxyphenyl) ketone, zinc powder, and TiCl4 was 1:(2~12):(1~2). The mixture was refluxed and stirred at 60 °C to 70 °C for 12 h to 30 h. After the reaction was completed, the reaction was terminated with Na2CO3 solution. The reaction product was extracted with dichloromethane and saturated NaCl solution to remove the solvent from the extracted product and purified to obtain a white solid, which was intermediate product 2. Step 2: Intermediate product 2, 1,2-bis(2-bromoethoxy)ethane, K2CO3 and KI were added to an acetone solution, wherein the molar ratio of intermediate product 2, 1,2-bis(2-bromoethoxy)ethane, K2CO3 and KI was 100:(400~600):(400~600):(4~6). The mixture was refluxed and stirred at 55 °C~60 °C for 12 h~24 h. After the reaction was completed, the mixture was purified to obtain a white solid, which was intermediate product 3. Step 3: In a nitrogen atmosphere, intermediate 3, 2,1,3-benzothiadiazole-4,7-bis(pinacol borate), tetra(triphenylphosphine)palladium, and K2CO3 aqueous solution were sequentially added to toluene. The molar ratio of intermediate 3, 2,1,3-benzothiadiazole-4,7-bis(pinacol borate), and tetra(triphenylphosphine)palladium was 100:(100~120):(3~10). The mixture was refluxed and stirred at 85 °C~95 °C for 24 h~36 h. After the reaction was completed, the mixture was dried with anhydrous magnesium sulfate and then purified to obtain an orange solid as intermediate 4. Step 4: Under a nitrogen atmosphere, intermediate product 4 was dissolved in anhydrous pyridine and refluxed at 95 °C to 105 °C for 48 h to 60 h. After the organic solvent was removed by rotary evaporation, the product was washed with acetone, filtered, and the precipitate was collected. After vacuum drying, an orange-red solid was obtained, which is the lysosome-targeted viscosity-responsive AIE polymer probe.

3. The method according to claim 2, characterized in that, Step 1 terminates the reaction with a 10% (w / w) Na2CO3 solution, with the volume ratio of anhydrous tetrahydrofuran to Na2CO3 solution being 10:(2~5).

4. The method according to claim 2, characterized in that, Step 2 purification was performed using column chromatography.

5. The method according to claim 2, characterized in that, In step 3, the concentration of the K2CO3 aqueous solution is 2 mol / L, and the amount used is the same as the volume of the toluene solution.

6. The method according to claim 2, characterized in that, The purification process described in step 3 involves purifying the residues obtained from the reaction using column chromatography.

7. The application of the lysosome-targeted viscosity-responsive AIE polymer probe PTPEB-Py as described in claim 1 in the preparation of a device for visualizing changes in lysosomal viscosity before and after drug treatment.

8. The application according to claim 7, characterized in that, The application visualizes changes in lysosomal viscosity during mitochondrial autophagy in cancer cells by constructing an intrinsic relationship between drug action time, lysosomal viscosity, and fluorescence lifetime.

9. A compound, characterized in that, The structural formula is as follows: 。 10. The use of the compound of claim 9 as a lysosomal targeted viscosity-responsive AIE polymer probe.

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