A mitochondria-targeting viscosity-responsive AIE probe, and a preparation method and application thereof

Abstract

The application discloses a mitochondrion-targeting viscosity-responsive AIE probe and a preparation method and application thereof. The AIE molecule is constructed by using TPE and TPP, an AIE probe molecule (TPE-4TPP) derived from TPE is designed, a drug action time-viscosity-fluorescence lifetime relationship diagram is established by a fluorescence lifetime signal, and a mitochondrion-targeting viscosity-responsive AIE probe with visualized mitochondrion viscosity change value is constructed. The "viscosity responsiveness" refers to that the fluorescence intensity and lifetime of the AIE probe are enhanced with the increase of viscosity, namely, the AIE probe can respond to the viscosity change of the environment. The mitochondrion-targeting viscosity-responsive AIE probe has the following molecular structure: the AIE probe has AIE property and mitochondrion targeting property, has good biocompatibility in cells, is a small-molecule probe with viscosity responsiveness to cell viscosity, and thus realizes the visualization of the mitochondrion viscosity change value in cancer cells through the fluorescence intensity and lifetime change.

Description

Technical Field

[0001] This invention belongs to the field of small molecules in materials medicine, specifically relating to the preparation of a viscosity-responsive AIE probe and its application in visualizing changes in mitochondrial viscosity in cancer cells. Background Technology

[0002] Mitochondria, vital organelles often referred to as the "powerhouse," have viscosity that is a key parameter for assessing cellular and mitochondrial dysfunction. In normal cells, the local viscosity of mitochondria can reach up to 100 mPa·s, far exceeding the viscosity of the cytoplasmic aqueous phase (1–2 mPa·s). Abnormal mitochondrial viscosity can lead to abnormal energy metabolism, increased apoptosis, and decreased immune function, resulting in various diseases such as malignant tumors, diabetes, liver damage, and Alzheimer's disease. After drug treatment, mitochondrial viscosity can spike to hundreds of mPa·s. Therefore, detecting changes in mitochondrial viscosity is crucial for understanding mitochondrial physiological activities and function, and for aiding in the diagnosis and treatment of related diseases.

[0003] Traditional viscosity measurement tools, including cup-type, vibratory, falling-ball, and capillary viscometers, are limited to measuring the viscosity of macroscopic liquid samples in vitro and cannot be used for intracellular microscopic viscosity measurement. Aggregation-induced emission (AIE) materials, due to their strong fluorescence properties in viscous environments, as well as their non-invasive labeling, high sensitivity, and high brightness, hold promise for intracellular, especially mitochondrial, viscosity measurement. For example, Ge et al. (JY Zhao, G. Zhang, HCHao, R. Sun, YJ Xu, JF Ge. Fluorescent probes based on quinoline and naphthidine derivatives with NIR and AIE properties for real-time monitoring mitochondrial viscosity during mitophagy. Sensor. Actuat. B. Chem., 2024, 401, 135010.) reported a class of mitochondrial-targeted near-infrared AIE probes for detecting viscosity changes during mitophagy; Zhu et al. (YDDou, Kenry, J. Liu, FF Zhang, CHCaia, Q. Zhu. 2-Styrylquinoline-based two-photon AIEgens for dual monitoring of pH and viscosity in A mitochondrial fluorescent probe (HAPH-1) sensitive to viscosity and pH was developed and used for viscosity and pH detection in live cells using two-photon microscopy. However, while these mitochondrial probes can reflect changes in mitochondrial viscosity through changes in fluorescence intensity, they cannot measure the viscosity value. Furthermore, the fluorescence intensity signal can be affected by non-viscosity-related factors such as photobleaching, probe concentration, and excitation light intensity. Unlike fluorescence intensity, fluorescence lifetime is not affected by fluorophore concentration, providing more stable and reliable data for monitoring viscosity changes in complex cellular environments. Therefore, there is an urgent need to develop a mitochondrial-targeted viscosity-responsive AIE probe to visualize specific changes in mitochondrial viscosity within cancer cells through changes in fluorescence lifetime. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing methods by providing a mitochondrial-targeted viscosity-responsive AIE probe, its preparation method, and its application. This invention yields a mitochondrial probe that possesses AIE properties and mitochondrial targeting, exhibits good biocompatibility within cells, and responds to cell viscosity. This allows for the visualization of mitochondrial viscosity changes within cancer cells by constructing the intrinsic relationship between drug action time, viscosity, and fluorescence lifetime (t-η-τ) through intracellular and extracellular fluorescence lifetime signals.

[0005] The concept of this invention is to develop a probe possessing AIE (Anaerobic Intracranial Effluent) properties and mitochondrial localization function, while introducing a positive charge to promote the effective accumulation of the AIE probe at the mitochondrial membrane potential. Tetraphenylphenylene (TPE), as a typical AIE molecule, has excellent structural designability; while triphenylphosphine (TPP) is an effective mitochondrial targeting site. Combining the two provides a new concept for creating a mitochondrial viscosity probe. This invention proposes to construct an AIE molecule using TPE and TPP, design a TPE-derived AIE probe molecule (TPE-4TPP), and visualize the mitochondrial viscosity change value by constructing a three-dimensional linear relationship of t-η-τ using intracellular and extracellular fluorescence lifetime signals to create a mitochondrial-targeted viscosity-responsive AIE probe.

[0006] The term "mitochondrial targeting" as used in this specification refers to the ability of molecules to specifically stain mitochondria after entering the cell.

[0007] 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.

[0008] In this invention, the reactants or organic solvents are all existing products that can be purchased from the market or made in-house.

[0009] The mitochondrial-targeted viscosity-responsive AIE probe provided by this invention has the following molecular structure:

[0010]

[0011] The preparation method of the above-mentioned mitochondrial-targeted viscosity-responsive AIE probe provided by the present invention includes the following synthesis process:

[0012]

[0013] The preparation method specifically includes the following steps:

[0014] Step 1:

[0015] TiCl4 solution was added to a mixture of 4,4′-dimethylbenzophenone, zinc powder, and THF (tetrahydrofuran) solution under low temperature (-78℃~0℃) and nitrogen atmosphere, wherein the molar ratio of 4,4′-dimethylbenzophenone, zinc powder, and TiCl4 was 1:(2~12):(1~2). The mixture was refluxed and stirred at 60℃~70℃ for 12h~30h. After the reaction was completed, the reaction was terminated with Na2CO3 solution. The product was extracted with dichloromethane (DCM) and saturated NaCl solution to remove the solvent from the extract and purified to obtain a white solid, which was intermediate product 2.

[0016] Step 2:

[0017] Intermediate product 2, N-bromosuccinimide, and benzoyl peroxide were dissolved in CCl4 solution, with the molar ratio of intermediate product 2, N-bromosuccinimide, and benzoyl peroxide being 100:(400-600):(5-8). The mixture was refluxed and stirred at 70-80°C for 12-30 hours under nitrogen atmosphere. After the reaction was completed, the mixture was quenched with saturated NaCl solution and DCM and purified to obtain a white solid, namely intermediate product 3.

[0018] Step 3:

[0019] Intermediate product 3 and triphenylphosphine were mixed with N,N-dimethylformamide, wherein the molar ratio of intermediate product 3 to triphenylphosphine was 1:(4-8). After stirring thoroughly at 90℃-120℃ for 18h-30h, the resulting mixture was subjected to solid-liquid separation and washing to obtain a white solid, which is the mitochondrial targeted viscosity-responsive AIE probe.

[0020] In the above method, step 1 is further terminated by using a 10% (w / w) Na₂CO₃ solution.

[0021] In the above method, further, step 1 purification is performed using column chromatography (DCM:PE = 1:2, v / v).

[0022] In the above method, further, the purification process described in step 2 is to purify the residue obtained from the reaction by column chromatography (PE:EtOAc = 10:1, v / v).

[0023] This invention also provides the application of the aforementioned mitochondrial-targeted viscosity-responsive AIE probe TPE-4TPP in visualizing changes in cellular mitochondrial viscosity. Preferably, this application visualizes changes in cancer cell mitochondrial viscosity by constructing an intrinsic relationship between drug action time, viscosity, and fluorescence lifetime (t-η-τ). During the visualization of mitochondrial viscosity changes in cancer cells, the AIE probe TPE-4TPP can visualize these changes through variations in fluorescence intensity and fluorescence lifetime. Therefore, the mitochondrial-targeted viscosity-responsive AIE probe described in this invention provides a potential research tool for mitochondrial physiology studies.

[0024] The present invention also provides a compound, characterized in that it has the following structural formula:

[0025]

[0026] This invention also provides the application of the above-mentioned compounds as mitochondrial-targeted viscosity-responsive AIE probes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The mitochondrial-targeted viscosity-responsive AIE probe of the present invention utilizes its AIE properties and water solubility to significantly enhance fluorescence intensity and lifetime when viscosity increases, possessing the potential to detect viscosity changes and can be applied to visualize changes in mitochondrial viscosity in cancer cells.

[0029] 2. The AIE probe of the present invention establishes a three-dimensional intrinsic relationship diagram of t-η-τ through intracellular and extracellular fluorescence lifetime signals to visualize the change value of mitochondrial viscosity, and this process is not affected by non-viscosity-related factors such as photobleaching, probe concentration and excitation light intensity.

[0030] 3. The mitochondrial-targeted viscosity-responsive AIE probe of the present invention is prepared by only three steps compared with traditional commercial mitochondrial dyes. The raw materials are widely available and the cost is low, which has great advantages in terms of ease of preparation and economy. Attached Figure Description

[0031] Figure 1 The image shows the 1H NMR spectrum of intermediate product 2 in Example 1.

[0032] Figure 2 The image shows the 1H NMR spectrum of intermediate product 3 in Example 1.

[0033] Figure 3 The 1H NMR spectrum of the mitochondrial-targeted viscosity-responsive AIE probe prepared in Example 1.

[0034] Figure 4The fluorescence spectra of TPE-4TPP in solid and DMSO solution states are shown.

[0035] Figure 5 The fluorescence spectra of TPE-4TPP at different viscosities and the established linear relationship between fluorescence growth factor and viscosity are shown.

[0036] Figure 6 The fluorescence lifetime of TPE-4TPP at different viscosities and the established linear relationship between fluorescence lifetime growth factor and viscosity are shown.

[0037] Figure 7 Cell viability after 2 hours of staining with different concentrations of TPE-4TPP.

[0038] Figure 8 Cell survival rates after treatment with different concentrations of dexamethasone for 12h, 24h, 36h and 48h.

[0039] Figure 9 This study investigated the co-localization of TPE-4TPP and Mito tracker green in leukemia cells.

[0040] Figure 10 The graph shows the linear relationship between the mean fluorescence intensity of cells as determined by flow cytometry and the duration of dexamethasone treatment.

[0041] Figure 11 The graph shows the linear relationship between mitochondrial fluorescence lifetime as measured by two-photon laser confocal microscopy and dexamethasone treatment time.

[0042] Figure 12 Fluorescence lifetime images for two-photon laser confocal microscopy before and 24 hours after dexamethasone treatment.

[0043] Figure 13 A three-dimensional linear relationship diagram of t-η-τ was established with fluorescence lifetime as the node between the inside and outside of the cell (in the figure, t is the drug action time, η is the viscosity, and τ is the fluorescence lifetime). Detailed Implementation

[0044] 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.

[0045] Example 1: Preparation of mitochondrial-targeted viscosity-responsive AIE probe:

[0046] Step 1

[0047] TiCl4 (4.95 mL, 45 mmol) was slowly added dropwise to a round-bottom flask containing 4,4′-dimethylbenzophenone (6.31 g, 30 mmol), zinc powder (5.88 g, 90 mmol), and THF solution (100 mL) under nitrogen atmosphere at 0 °C. The mixture was stirred at 64 °C for 18 h, and the reaction was terminated with 10% Na2CO3 solution. The product was extracted with dichloromethane (DCM) and saturated NaCl solution, and the solvent was removed by rotary evaporation. Finally, the product was purified by column chromatography (DCM:PE = 1:2, v / v) to give intermediate 2 as a white solid (5.54 g, 95% yield). 1 H NMR (600MHz, CDCl3, δ, Figure 1 ): 6.90 (s, 16H, Ar-H), 2.26 (s, 12H, -CH3).

[0048] Step 2

[0049] Intermediate product 2 (5.54 g, 14.25 mmol), N-bromosuccinimide (11.16 g, 62.70 mmol), and benzoyl peroxide (0.21 g, 0.86 mmol) were placed in a 200 mL CCl4 reaction flask. The mixture was stirred at 80 °C for 18 h under nitrogen atmosphere. The mixture was then quenched with saturated NaCl solution and DCM. The residue was purified by column chromatography (PE:EtOAc = 10:1, v / v) to give intermediate product 3 as a white solid (5.62 g, 56% yield). 1 H NMR (600MHz, CDCl3, δ, Figure 2 ): 7.14–7.13 (d, 8H, Ar-H), 6.96–6.95 (d, 8H, Ar-H), 4.42 (s, 8H, -CH2-).

[0050] Step 3

[0051] Intermediate product 3 (5.62 g, 7.98 mmol) and triphenylphosphine (16.74 g, 63.84 mmol) were added to a round-bottom flask containing N,N-dimethylformamide (300 mL), and the mixture was magnetically stirred at 100 °C for 24 h. After the reaction was complete, the mixture was centrifuged and washed with a large amount of toluene to obtain the target product TPE-4TPP as a white solid (11.19 g, 80% yield). 1 H NMR (600MHz, DMSO, δ, Figure 3): 7.91–7.89 (t, 12H, Ar-H), 7.73–7.70 (m, 24H, Ar-H), 7.65–7.62 (m, 24H, Ar-H ), 6.78–6.76 (d, 8H, Ar-H), 6.72–6.70 (d, 8H, Ar-H), 5.14–5.11 (d, 5H, -CH2-).

[0052] Example 2: Preparation of mitochondrial-targeted viscosity-responsive AIE probe:

[0053] Step 1

[0054] TiCl4 (2.20 mL, 20 mmol) was slowly added to a round-bottom flask containing 4,4′-dimethylbenzophenone (2.10 g, 10 mmol), zinc powder (6.53 g, 100 mmol), and THF solution (600 mL) at –78 °C under nitrogen atmosphere. The mixture was stirred at 70 °C for 30 h, and the reaction was terminated with 10% Na2CO3 solution. The product was extracted with DCM and saturated NaCl solution, and the solvent was removed by rotary evaporation. Purification was performed by column chromatography (DCM:PE = 1:2, v / v) to give a white solid intermediate 2 (1.75 g, 90% yield).

[0055] Step 2

[0056] Intermediate 2 (1.75 g, 4.50 mmol), N-bromosuccinimide (4.81 g, 27.00 mmol), and benzoyl peroxide (0.09 g, 0.36 mmol) were added to a reaction flask containing 100 mL of CCl4. The mixture was stirred at 70 °C for 30 h under nitrogen atmosphere. The reaction was quenched with saturated NaCl solution and DCM. Purification by column chromatography (PE:EtOAc = 10:1, v / v) gave a white solid intermediate 3 (1.90 g, 60% yield).

[0057] Step 3

[0058] Intermediate product 3 (1.90 g, 2.70 mmol) and triphenylphosphine (2.83 g, 10.80 mmol) were added to a round-bottom flask containing N,N-dimethylformamide (100 mL), and the mixture was magnetically stirred at 120 °C for 30 h. After the reaction was complete, the mixture was centrifuged and washed with a large amount of toluene to obtain the white solid target product TPE-4TPP (3.31 g, 70% yield).

[0059] Example 3

[0060] This embodiment measures the AIE properties of the TPE-4TPP prepared according to the present invention.

[0061] Test method: TPE-4TPP was dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.2 mM solution. The fluorescence spectra of TPE-4TPP in solid and solution states were measured using a HORIBA FluoroMax-4 fluorescence spectrometer. The results are as follows: Figure 4 As shown.

[0062] Results and Analysis: From Figure 4 It can be seen that TPE-4TPP exhibits almost no fluorescence in DMSO solution, where intramolecular motion is relatively free, and excited-state energy is depleted through non-radiative pathways. However, in the solid state, TPE-4TPP emits strong fluorescence due to restricted intramolecular motion. These experimental results demonstrate that the TPE-4TPP molecule synthesized in this invention possesses typical AIE properties.

[0063] Example 4

[0064] This embodiment measures the viscosity response of the TPE-4TPP prepared according to the present invention.

[0065] Test method: TPE-4TPP was dissolved in polyethylene oxide (M) of different viscosities. w In a TPE-4TPP solution with a molar concentration of 0.2 mM and a TPE-4 water mixture of 8 million TPEs, the fluorescence intensity of the solution at different viscosities was measured using a HORIBA FluoroMax-4 fluorescence spectrometer. A linear relationship between relative fluorescence intensity and viscosity was plotted, and the results are as follows: Figure 5 As shown.

[0066] Results and Analysis: From Figure 5 It can be seen that the fluorescence of TPE-4TPP is very weak at a low viscosity of 16.93 mPa·s; however, the fluorescence signal gradually increases with increasing viscosity, and even at a viscosity of 680.69 mPa·s, the fluorescence intensity increases by about 12 times. The fluorescence intensity of TPE-4TPP exhibits a good linear relationship with the viscosity of its environment, R0 2 =0.99. This experimental result indicates that the fluorescence intensity of the AIE molecule TPE-4TPP synthesized in this invention is highly responsive to viscosity.

[0067] Example 5

[0068] This embodiment measures the fluorescence lifetime of the TPE-4TPP prepared in this invention at different viscosities.

[0069] Test method: TPE-4TPP was dissolved in polyethylene oxide (M) of different viscosities. wIn a TPE-4TPP solution with a molar concentration of 0.2 mM and a TPE-water mixture of 8 million TPEs, the fluorescence lifetime of the solution at different viscosities was measured using a Fluorolog-3 steady-state-transient near-infrared fluorescence microscopy spectrometer. A linear relationship between fluorescence lifetime and viscosity was plotted, and the results are as follows: Figure 6 As shown.

[0070] Results and Analysis: From Figure 6 It can be seen that at a low viscosity of 3.26 mPa·s, the fluorescence lifetime of TPE-4TPP is low, approximately 0.219 ns; as the viscosity increases, the fluorescence lifetime gradually increases. The fluorescence lifetime of TPE-4TPP exhibits a good linear relationship with the viscosity of the environment, indicating that the fluorescence lifetime of the synthesized AIE molecule TPE-4TPP in this invention shows a one-to-one correspondence with viscosity in different viscosity environments, and the fluorescence lifetime is highly responsive to viscosity.

[0071] Example 6

[0072] This embodiment measures the cytotoxicity of the TPE-4TPP prepared according to the present invention.

[0073] 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 / mL. Next, TPE-4TPP was dissolved in sterile DMSO solution and added to the suspended cells to form working concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM, respectively. Finally, cell viability was determined using the CCK-8 assay to assess the potential toxicity of the AIE probe.

[0074] Results and Analysis: From Figure 7 It can be seen that when the concentration of TPE-4TPP is increased to 60 μM, the cell survival rate is still as high as 90%, indicating that TPE-4TPP has low cytotoxicity to living cells and good biocompatibility.

[0075] Example 7

[0076] This embodiment measures the cytotoxicity of dexamethasone (an anticancer drug).

[0077] Test method: ① Preparation of cell suspension: Leukemia cells were taken, suspended in culture medium, and the cell concentration was adjusted to 1.0 × 10⁻⁶. 6 / mL. ② Drug preparation: Dexamethasone was dissolved in sterile DMSO solution and then added to the cell suspension to make final concentrations of 0.25μM, 0.50μM, 0.75μM, 1.00μM, 1.25μM, and 1.50μM, respectively. ③ Cell culture: The above cell suspensions were divided into four groups and cultured in an incubator at 37℃ and 5% CO2 for 12h, 24h, 36h, and 48h, respectively. ④ Cell viability assay: The CCK-8 assay was used to evaluate cell viability at different drug treatment times and concentrations to analyze the cytotoxic effect of dexamethasone.

[0078] Results and Analysis: Figure 8 As shown, a slight decreasing trend in cell viability was observed with increasing dexamethasone concentration. However, a more significant change was the substantial decrease in cell viability with prolonged dexamethasone treatment time. Specifically, after 24 hours of drug treatment, the cell viability was approximately 80%; however, when the treatment time was extended to 48 hours, the cell viability dropped sharply to only 15%. This data indicates that the cytotoxic effect of dexamethasone significantly increases with time.

[0079] Example 8

[0080] This embodiment measures the colocalization of the TPE-4TPP prepared in this invention with a commercial probe.

[0081] Test method: First, leukemia cells were taken, suspended in culture medium, and the cell concentration was adjusted to 1.0 × 10⁻⁶. 6 / mL. Next, TPE-4TPP (final working concentration 50 μM) was added to the cell suspension and incubated at 37°C and 5% CO2 for 2 h. Then, the commercial mitochondrial targeting dye Mito tracker green (final working concentration 250 nM) was added, and incubation continued for 30 min. Following this, the cells were washed three times by centrifugation with phosphate-buffered saline to remove excess dye and culture medium. Finally, imaging was performed using an Olympus FV3000 laser confocal microscope. The excitation wavelength for the blue channel was set to 405 nm, and the collection wavelength range was 430–470 nm; the excitation wavelength for the green channel was set to 488 nm, and the collection wavelength range was 500–550 nm. These steps effectively stain and image leukemia cells to observe the distribution of TPE-4TPP and commercial mitochondrial dyes within the cells.

[0082] Results and Analysis: Figure 9As shown, the left image is a fluorescence image of TPE-4TPP, the middle image is a fluorescence image of Mito trackergreen, and the right image is a fluorescence image of both co-stained. The results show that the fluorescence signal overlap coefficient between the fluorescent probe of this invention and commercial mitochondrial dyes in cells is as high as 0.80, indicating that the fluorescent probe can locate mitochondria, demonstrating the mitochondrial targeting capability of the fluorescent probe.

[0083] Example 9

[0084] This embodiment tests the intracellular viscosity response of TPE-4TPP prepared in this invention.

[0085] Experiment 1

[0086] Test method: First, the concentration of leukemia cells in the culture medium was adjusted to 1.0 × 10⁻⁶. 6 / mL. Next, dexamethasone was added to the cell suspension to achieve a final working concentration of 1.00 μM, and 18 mL of cells were divided into 9 groups and incubated at 37°C, 5% CO2 for 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 21 h, and 24 h, respectively. Then, TPE-4TPP was added to achieve a final working concentration of 50 μM, and staining was performed for 2 h. Finally, fluorescence intensity was measured using a BD Celesta flow cytometer to observe changes in mitochondrial viscosity within the cells.

[0087] Results and Analysis: To clearly observe the change in fluorescence intensity over time, we plotted a graph showing the relationship between fluorescence intensity and drug action time. (See figure below.) Figure 10 As shown, the fluorescence intensity increased linearly with increasing dexamethasone treatment time. This indicates that after drug induction, mitochondrial viscosity increased linearly with prolonged treatment time.

[0088] Experiment 2

[0089] Test method: First, the concentration of leukemia cells in the culture medium was adjusted to 1.0 × 10⁻⁶. 6 / mL. Then, 14mL of cell suspension was divided into 7 groups and dexamethasone was added to achieve a final working concentration of 1.00μM. The cells were incubated at 37℃, 5% CO2 for 0h, 4h, 8h, 12h, 16h, 20h, and 24h, respectively. After incubation, TPE-4TPP (final working concentration 50μM) was added to the cells for staining for 2h. Finally, N-SIM / A1R MP was used. + Fluorescence lifetime imaging was tested using a high-resolution two-photon laser confocal microscope.

[0090] Results and Analysis: Figure 11As shown, to clearly observe the change in fluorescence lifetime with different treatment times of dexamethasone, we plotted the relationship between fluorescence lifetime and time. The fluorescence lifetime showed a linear increase with increasing drug treatment time. This indicates that after drug induction, mitochondrial viscosity increases linearly with increasing drug treatment time. Figure 12 As shown, before dexamethasone treatment, the fluorescence lifetime of mitochondria in leukemia cells was approximately 500 ps; after 24 h of dexamethasone treatment, the fluorescence lifetime increased to 950 ps. Based on the previously established linear relationship between fluorescence lifetime and viscosity in the extracellular environment, it can be inferred that the mitochondrial viscosity increased from 60 mPa·s to approximately 600 mPa·s. Using fluorescence lifetime as the connecting point, a three-dimensional linear relationship graph of t-η-τ was constructed by combining the τ-η (fluorescence lifetime-viscosity) relationship established in the extracellular environment with the τ-t (fluorescence lifetime-drug action time) linear relationship established in the intracellular environment to visualize the change in mitochondrial viscosity. Figure 13 As shown, both the viscosity and fluorescence lifetime of intracellular mitochondria increased with prolonged drug action time. The fluorescence lifetime increased linearly with increasing viscosity, meaning that changes in mitochondrial viscosity can be visualized using fluorescence lifetime. This result validates that the TPE-4TPP probe can monitor and visualize changes in mitochondrial viscosity through variations in its fluorescence lifetime.

Claims

1. A compound, characterized in that, The molecular structure is as follows: 。 2. A process for the preparation of a compound according to claim 1, characterized in that, The method comprises the following steps: Step 1: A mixture of 4,4'-dimethylbenzophenone, zinc powder and tetrahydrofuran solution is added with TiCl4 solution under the conditions of-78 ℃~0 ℃ and nitrogen environment, wherein the molar ratio of 4,4'-dimethylbenzophenone, zinc powder and TiCl4 is 1:(2~12):(1~2) in sequence, and the mixture is stirred at 60 ℃~70 ℃ for 12 h~30 h; the reaction is terminated with Na2CO3 solution, the product is extracted with dichloromethane and saturated NaCl solution, the solvent in the extracted product is removed, and a white solid is obtained by purification, which is an intermediate product 2, and the structural formula is as follows ； Step 2: The intermediate product 2, N-bromosuccinimide and dibenzoyl peroxide are dissolved in a CCl4 solution, wherein the molar ratio of the intermediate product 2, N-bromosuccinimide and dibenzoyl peroxide is 100:(400~600):(5~8) in sequence, and the mixture is stirred at 70 ℃~80 ℃ under the conditions of nitrogen environment and reflux for 12 h~30 h; after the reaction is completed, the mixture is quenched with saturated NaCl solution and DCM, and the white solid obtained by purification treatment is an intermediate product 3, and the structural formula is as follows ； Step 3: The intermediate product 3 and triphenylphosphine are mixed with N,N-dimethylformamide, wherein the molar ratio of the intermediate product 3 and triphenylphosphine is 1:(4~8), and the mixture is fully stirred at 90 ℃~120 ℃ for 18 h~30 h; the mixture obtained by the reaction is subjected to solid-liquid separation and washing to obtain a white solid, which is a mitochondria-targeting viscosity-responsive AIE probe.

3. The method of claim 2, wherein, In step 1, the reaction is terminated with a 10% mass concentration Na2CO3 solution.

4. The method of claim 2, wherein, In step 1, the purification is performed by column chromatography.

5. The method of claim 2, wherein, In step 2, the purification treatment is performed by column chromatography.

6. Use of the compound in claim 1 in the preparation of a probe for visualizing changes in the viscosity of cell mitochondria.

7. Use according to claim 6, characterized in that, The application is a probe for preparing visualization of cancer cell mitochondrial viscosity changes by constructing the drug action time-viscosity-fluorescence lifetime ( ) intrinsic relationship.

8. Use of a compound in the preparation of a mitochondria-targeting viscosity-responsive AIE probe, and the structural formula of the compound is as follows, 。

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