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

Abstract

The invention discloses a mitochondrial targeting viscosity response AIE probe as well as a preparation method and application thereof. According to the present invention, TPE and TPP are adopted to construct an AIE molecule, a TPE-derived AIE probe molecule (TPE-4TPP) is designed, a drug action time-viscosity-fluorescence lifetime relationship diagram is established through a fluorescence lifetime signal, and the mitochondrial targeting viscosity response AIE probe with a visual mitochondrial viscosity change value is constructed. The viscosity responsiveness means that the fluorescence intensity and the service life of the AIE probe are enhanced along with the increase of the viscosity, that is, the AIE probe can respond to the viscosity change of the environment where the AIE probe is located. The molecular structure of the mitochondrial targeting viscosity response AIE probe is as follows: # imgabs0 #, and the mitochondrial targeting viscosity response AIE probe has AIE properties and mitochondrial targeting, has good biocompatibility in cells and responsiveness to cell viscosity, so that the change value of the mitochondrial viscosity in cancer cells is visualized through the change of fluorescence intensity and service life.

Description

Technical Field

[0001] The present invention belongs to the field of small molecules in material medicine, and specifically relates to the preparation of a viscosity-responsive AIE probe and its application in the field of visualizing changes in viscosity of cancer cell mitochondria. Background Art

[0002] Mitochondria, an important organelle known as the "powerhouse", its viscosity is a key parameter for evaluating cell and mitochondrial dysfunction. In normal cells, the local viscosity of mitochondria is as high as 100mPa·s, which is much greater than the viscosity of the cytoplasmic aqueous phase (1-2mPa·s). Abnormal mitochondrial viscosity may lead to abnormal energy metabolism, increased cell apoptosis and decreased immune function, which in turn may cause a variety of diseases such as cell malignancies, diabetes, liver damage, Alzheimer's disease, etc. After drug treatment, mitochondrial viscosity can soar to hundreds of mPa·s. Therefore, detecting changes in mitochondrial viscosity is crucial for understanding mitochondrial physiological activities and functions and assisting in the diagnosis and treatment of related diseases.

[0003] Traditional viscosity detection tools, including cup, vibration, falling ball and capillary viscometers, are limited to the measurement of macroscopic liquid sample viscosity in vitro and cannot be used for microscopic viscosity detection in cells. Aggregation-induced emission (AIE) materials are expected to be used for intracellular viscosity detection, especially mitochondrial viscosity detection, due to their strong fluorescence properties in a viscous environment, and their non-invasive labeling, high sensitivity and high brightness. For example, Ge et al. (JYZhao, G.Zhang, HCHao, R.Sun, YJXu, JFGe. Fluorescent probes based on quinoline and naphthidine derivatives with NIR and AIE properties for real-time monitoringmitochondrial 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 mitochondrial autophagy; Zhu et al. (YDDou, Kenry, J.Liu, FFZhang, CHCaia, Q.Zhu. 2-Styrylquinoline-based two-photon AIEgens for dual monitoring of pH and viscosity in livingcells.J.Mater.Chem.B,2019,7,7771-7775.) developed an AIE fluorescent probe (HAPH-1) that is sensitive to viscosity and pH, and used it for viscosity and pH detection in living cells using two-photon microscopy. However, although these mitochondrial probes can reflect changes in mitochondrial viscosity through changes in fluorescence intensity, they cannot measure their viscosity values, and the fluorescence intensity signal may also 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, which provides 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, hoping to visualize the specific changes in mitochondrial viscosity in cancer cells through changes in fluorescence lifetime. Summary of the invention

[0004] The purpose of the present invention is to address the shortcomings of existing methods and provide a mitochondrial targeted viscosity-responsive AIE probe and its preparation method and application, so as to obtain a mitochondrial probe with AIE properties and mitochondrial targeting, good biocompatibility in cells, and responsiveness to cell viscosity, thereby realizing the visualization of the mitochondrial viscosity change value in cancer cells by constructing the intrinsic relationship of drug action time-viscosity-fluorescence lifetime (t-η-τ) through fluorescence lifetime signals inside and outside cells.

[0005] The idea of ​​the present invention is to develop a probe with AIE characteristics and mitochondrial localization function, while introducing positive charges to promote the effective accumulation of AIE probes on mitochondrial membrane potential. Tetraphenylethylene (TPE), as a typical AIE molecule, has excellent structural designability; and triphenylphosphine (TPP) is an effective mitochondrial targeting site. Combining the two provides a new concept for creating mitochondrial viscosity probes. The present invention proposes to use TPE and TPP to construct AIE molecules, design a TPE-derived AIE probe molecule (TPE-4TPP), and construct a mitochondrial-targeted viscosity-responsive AIE probe that visualizes the mitochondrial viscosity change value by constructing a t-η-τ three-dimensional linear relationship through intracellular and extracellular fluorescence lifetime signals.

[0006] The "mitochondrial targeting" mentioned in this specification refers to the ability of a molecule to specifically stain mitochondria after entering cells.

[0007] The “viscosity responsiveness” mentioned in this specification means that as the viscosity increases, the fluorescence intensity and lifetime of the AIE probe increase, that is, the AIE probe can respond to changes in the viscosity of the environment in which it is located.

[0008] In the present invention, the reactants or organic solvents are all existing products and can be purchased from the market or prepared by oneself.

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

[0010]

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

[0012]

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

[0014] Step 1:

[0015] Under low temperature (-78°C to 0°C) and nitrogen environment, a TiCl4 solution is added to a mixture of 4,4′-dimethylbenzophenone, zinc powder and THF (tetrahydrofuran) solution, wherein the molar ratio of 4,4′-dimethylbenzophenone, zinc powder and TiCl4 is 1:(2-12):(1-2), respectively, and the mixture is refluxed and stirred at 60°C to 70°C for 12h to 30h. After the reaction is completed, the reaction is terminated with a Na2CO3 solution; the product is extracted with dichloromethane (DCM) and a saturated NaCl solution, the solvent in the extracted product is removed, and the white solid is purified to obtain the intermediate product 2.

[0016] Step 2:

[0017] 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), respectively. The mixture is refluxed and stirred at 70°C-80°C for 12h-30h in a nitrogen environment. After the reaction is completed, the mixture is quenched with a saturated NaCl solution and DCM, and purified to obtain a white solid, i.e., the intermediate product 3.

[0018] Step 3:

[0019] The intermediate product 3 and triphenylphosphine are mixed with N,N-dimethylformamide, wherein the molar ratio of the intermediate product 3 to triphenylphosphine is 1:(4-8). After being fully stirred at 90°C-120°C for 18h-30h, the reaction mixture is subjected to solid-liquid separation and washed to obtain a white solid, which is a mitochondrial-targeted viscosity-responsive AIE probe.

[0020] In the above method, further, in step 1, a Na2CO3 solution with a mass concentration of 10% is used to terminate the reaction.

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

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

[0023] The present invention also provides the application of the above-mentioned mitochondrial targeted viscosity-responsive AIE probe TPE-4TPP in visualizing changes in cell mitochondrial viscosity. Preferably, the application is to visualize changes in mitochondrial viscosity of cancer cells by constructing the intrinsic relationship of drug action time-viscosity-fluorescence lifetime (t-η-τ). In the process of visualizing changes in mitochondrial viscosity of cancer cells, the AIE probe TPE-4TPP can visualize changes in mitochondrial viscosity through changes in fluorescence intensity and fluorescence lifetime. Therefore, the mitochondrial targeted viscosity-responsive AIE probe described in the present invention provides a potential research tool for the study of mitochondrial physiology.

[0024] The present invention also provides a compound, characterized in that the structural formula is as follows:

[0025]

[0026] The present invention also provides the use of the above compound as a mitochondrial targeted viscosity-responsive AIE probe.

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

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

[0029] 2. The AIE probe described in the present invention establishes a t-η-τ three-dimensional intrinsic relationship diagram through the fluorescence lifetime signals inside and outside the cell 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. Compared with traditional mitochondrial commercial dyes, the mitochondrial targeted viscosity-responsive AIE probe described in the present invention is prepared through only three steps of synthesis, has a wide source of raw materials, and is low in cost, and has great advantages in both ease of preparation and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate product 2 in Example 1.

[0032] Figure 2 The hydrogen nuclear magnetic resonance spectrum of the intermediate product 3 in Example 1 is shown in FIG.

[0033] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the mitochondrial targeted viscosity-responsive AIE probe prepared in Example 1.

[0034] Figure 4Fluorescence spectra of TPE-4TPP in solid state and DMSO solution state.

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

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

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

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

[0039] Fig. 9 This is a co-localization experiment of TPE-4TPP and Mito tracker green in leukemia cells.

[0040] Fig.10 This is a linear relationship diagram between the mean fluorescence intensity of cells measured by flow cytometry and the action time of dexamethasone.

[0041] Fig.11 This is the linear relationship between the fluorescence lifetime of mitochondria measured by two-photon laser confocal microscopy and the dexamethasone action time.

[0042] Fig.12 These are fluorescence lifetime images measured by two-photon laser confocal microscopy before and 24 hours after the action of dexamethasone.

[0043] Fig.13 This is a t-η-τ three-dimensional linear relationship diagram established with fluorescence lifetime as the node inside and outside the cell (in the figure, t is the drug action time, η is the viscosity, and τ is the fluorescence lifetime). DETAILED DESCRIPTION

[0044] The present invention is further described below by way of examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention according to the above-mentioned invention content and implement the invention specifically, which still falls 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) at 0 ° C and nitrogen environment. The mixture was stirred at 64 ° C for 18 h, and then 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, column chromatography (DCM: PE = 1: 2, v / v) was used to purify the intermediate product 2 as a white solid (5.54 g, yield 95%). 1 H NMR (600 MHz, CDCl3, δ, Figure 1 ): 6.90 (s, 16H, Ar-H), 2.26 (s, 12H, -CH3).

[0048] Step 2

[0049] Intermediate product 2 (5.54g, 14.25mmol), N-bromosuccinimide (11.16g, 62.70mmol) and dibenzoyl peroxide (0.21g, 0.86mmol) are placed in a reaction bottle of 200mL CCl4. Under nitrogen environment, this mixture is stirred at 80 ° C for 18h. Afterwards, quenched with saturated NaCl solution and DCM. Purification of the obtained residue by column chromatography (PE: EtOAc = 10: 1, v / v) gives intermediate product 3 as a white solid (5.62g, yield 56%). 1 H NMR (600 MHz, 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] The 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 magnetically stirred at 100°C for 24 h. After the reaction was completed, the mixture was poured into a centrifuge tube for centrifugation, and the obtained solid was washed with a large amount of toluene to finally obtain the target product TPE-4TPP as a white solid (11.19 g, yield 80%). 1 H NMR (600 MHz, 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 and nitrogen. After the mixture was stirred at 70°C for 30 h, 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 evaporator. Column chromatography purification (DCM: PE = 1: 2, v / v) gave a white solid intermediate 2 (1.75 g, yield 90%).

[0055] Step 2

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

[0057] Step 3

[0058] The 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 magnetically stirred at 120°C for 30 h. After the reaction was completed, the mixture was poured into a centrifuge tube for centrifugation, and the solid was washed with a large amount of toluene to obtain the target product TPE-4TPP (3.31 g, yield 70%) as a white solid.

[0059] Example 3

[0060] This example measures the AIE properties of TPE-4TPP prepared in the present invention.

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

[0062] Results and analysis: From Figure 4 It can be seen that in the DMSO solution state, TPE-4TPP has almost no fluorescence, at which point the intramolecular motion is relatively free and the excited state energy is depleted through non-radiative pathways; whereas in the solid state, TPE-4TPP emits strong fluorescence due to the restriction of intramolecular motion. This experimental result shows that the TPE-4TPP molecule synthesized by the present invention has typical AIE properties.

[0063] Example 4

[0064] This example measures the viscosity response of TPE-4TPP prepared in the present invention.

[0065] Test method: Dissolve TPE-4TPP in polyethylene oxide (M w =8 million)-water mixed solution, the molar concentration is 0.2mM, the fluorescence intensity of TPE-4TPP solution at different viscosities is tested using HORIBA FluoroMax-4 fluorescence spectrometer, and the linear relationship between relative fluorescence intensity and viscosity is plotted. The results are as follows Figure 5 shown.

[0066] Results and analysis: From Figure 5 It can be seen that at a low viscosity of 16.93 mPa·s, the fluorescence of TPE-4TPP is very weak; as the viscosity increases, the fluorescence signal gradually increases, and even when the viscosity is 680.69 mPa·s, the fluorescence increases by about 12 times. The fluorescence intensity of TPE-4TPP has a good linear relationship with the viscosity of the environment, R 2 =0.99. The experimental results show that the fluorescence intensity of the AIE molecule TPE-4TPP synthesized by the present invention is highly responsive to viscosity.

[0067] Example 5

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

[0069] Test method: Dissolve TPE-4TPP in polyethylene oxide (M w=8 million)-water mixed solution with a molar concentration of 0.2 mM. The fluorescence lifetime of TPE-4TPP solution at different viscosities was tested using a Fluorolog-3 steady-state-transient near-infrared microfluorescence spectrometer, and the linear relationship between fluorescence lifetime and viscosity was plotted. The results are shown in Figure 6 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, about 0.219 ns; as the viscosity increases, the fluorescence lifetime gradually increases. The fluorescence lifetime of TPE-4TPP has a good linear relationship with the viscosity of the environment in which it is located, indicating that the fluorescence lifetime of the AIE molecule TPE-4TPP synthesized in the present invention can show a one-to-one correspondence with the viscosity in different viscosity environments, and the fluorescence lifetime is highly responsive to the viscosity.

[0071] Example 6

[0072] This example measures the cytotoxicity of TPE-4TPP prepared in the present invention.

[0073] Test method: First, prepare a cell suspension, suspend leukemia cells in culture medium, and adjust the cell concentration to 1.0×10 6 / mL. Then, TPE-4TPP was dissolved in sterile DMSO solution and added to the suspended cells to form working concentrations of 10μM, 20μM, 30μM, 40μM, 50μM and 60μM, respectively. Finally, the CCK-8 method was used to determine the cell viability to evaluate 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 increased to 60 μM, the cell viability was still as high as 90%, indicating that TPE-4TPP had low cytotoxicity to living cells and had good biocompatibility.

[0075] Example 7

[0076] This example measures the cytotoxicity of dexamethasone, an anticancer drug.

[0077] Test method: ① Preparation of cell suspension: Take leukemia cells, suspend them in culture medium, and adjust the cell concentration to 1.0×10 6 / mL. ② Drug preparation: Dexamethasone was dissolved in sterile DMSO solution and then added to the cell suspension to make the final concentrations 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°C and 5% CO2 for 12h, 24h, 36h and 48h, respectively. ④ Cell viability detection: The CCK-8 method was used to evaluate the cell viability at different drug action times and concentrations to analyze the cytotoxic effect of dexamethasone.

[0078] Results and analysis: Figure 8 As shown, a slight downward trend in cell survival was observed with increasing dexamethasone concentrations. However, a more significant change was that cell survival decreased significantly with the extension of dexamethasone action time. Specifically, after 24 hours of drug action, cell survival was approximately 80%; and when the action time was extended to 48 hours, cell survival dropped sharply to only 15%. This data indicates that the cytotoxic effect of dexamethasone significantly increases with time.

[0079] Example 8

[0080] This example measures the co-localization of TPE-4TPP prepared by the present invention and commercial probes.

[0081] Test method: First, take leukemia cells, suspend them in culture medium, and adjust the cell concentration to 1.0×10 6 / mL. Secondly, TPE-4TPP (final working concentration of 50 μM) was added to the cell suspension and incubated in an incubator at 37°C and 5% CO2 for 2 hours. Subsequently, the commercial mitochondrial localization dye Mito tracker green (final working concentration of 250 nM) was added and incubated for another 30 minutes. Next, the cells were centrifuged and washed three times with phosphate buffer to remove excess dye and culture medium. Finally, an Olympus FV3000 laser confocal microscope was used for imaging. The excitation wavelength of the blue channel was set to 405 nm, and the collection wavelength range was 430 to 470 nm; the excitation wavelength of the green channel was set to 488 nm, and the collection wavelength range was 500 to 550 nm. Through the above steps, leukemia cells can be effectively stained and imaged to observe the distribution of TPE-4TPP and commercial mitochondrial dyes in cells.

[0082] Results and analysis: Fig. 9As shown, the left picture is the fluorescence imaging of TPE-4TPP, the middle picture is the fluorescence imaging of Mito trackergreen, and the right picture is the fluorescence image of the co-staining of the two. The results show that the fluorescence signal overlap coefficient of the fluorescent probe of the present invention and the commercial mitochondrial dye in cells is as high as 0.80, indicating that the fluorescent probe can locate mitochondria, proving the mitochondrial targeting of the fluorescent probe.

[0083] Example 9

[0084] This example tests the viscosity response of TPE-4TPP prepared by the present invention in cells.

[0085] Experiment 1

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

[0087] Results and analysis: In order to clearly observe the law of fluorescence intensity changing with time, we plotted the relationship between fluorescence intensity and drug action time. Fig.10 As shown in the figure, as the action time of dexamethasone increases, the fluorescence intensity shows a linear increase trend. This indicates that after drug induction, the viscosity of mitochondria increases linearly with the extension of action time.

[0088] Experiment 2

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

[0090] Results and analysis: Fig.11As shown in the figure, in order to clearly observe the change of fluorescence lifetime with different action time of dexamethasone, we plotted the relationship between fluorescence lifetime and time. As the drug action time increases, the fluorescence lifetime increases linearly. This shows that after drug induction, the viscosity of mitochondria increases linearly with the extension of drug action time. Fig.12 As shown in the figure, before the action of dexamethasone, the fluorescence lifetime of leukemia cell mitochondria was about 500ps; after 24 hours of dexamethasone treatment, the fluorescence lifetime was extended to 950ps. Based on the linear relationship between fluorescence lifetime and viscosity established extracellularly before, it can be inferred that the viscosity of mitochondria increased from 60mPa·s to about 600mPa·s. Taking the fluorescence lifetime as the connection point, a t-η-τ three-dimensional linear relationship diagram is constructed by combining the τ-η (fluorescence lifetime-viscosity) relationship established extracellularly and the τ-t (fluorescence lifetime-drug action time) linear relationship established inside the cell to visualize the change in mitochondrial viscosity. As shown Fig.13 As shown in the figure, as the drug action time increases, the viscosity and fluorescence lifetime of the mitochondria in the cells show an increasing trend. As the viscosity increases, the fluorescence lifetime increases linearly, and the change in mitochondrial viscosity can be visualized by the fluorescence lifetime. This result verifies that the TPE-4TPP probe can monitor and visualize changes in mitochondrial viscosity through changes in its fluorescence lifetime.

Claims

1. A mitochondrial-targeted viscosity-responsive AIE probe, characterized in that: Its molecular structure is as follows:

2. The method for preparing the mitochondrial targeted viscosity-responsive AIE probe according to claim 1, characterized in that: The following steps are involved: Step 1: Under a nitrogen environment of -78°C to 0°C, a TiCl4 solution is added to a mixture of 4,4′-dimethylbenzophenone, zinc powder and tetrahydrofuran solution, wherein the molar ratio of 4,4′-dimethylbenzophenone, zinc powder and TiCl4 is 1:(2-12):(1-2), and the mixture is refluxed and stirred at 60°C to 70°C for 12h to 30h; the reaction is terminated with a Na2CO3 solution, the product is extracted with dichloromethane and a saturated NaCl solution, the solvent in the extracted product is removed, and a white solid is purified to obtain the intermediate product 2; 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), respectively, and refluxed and stirred for 12h-30h at 70°C-80°C in a nitrogen environment. After the reaction is completed, quenching is performed with a saturated NaCl solution and DCM, and purification is performed to obtain a white solid, namely the intermediate product 3; Step 3: The intermediate product 3 and triphenylphosphine are mixed with N,N-dimethylformamide, wherein the molar ratio of the intermediate product 3 to triphenylphosphine is 1:(4-8). After being fully stirred at 90°C-120°C for 18h-30h, the reaction mixture is subjected to solid-liquid separation and washed to obtain a white solid, which is a mitochondrial-targeted viscosity-responsive AIE probe.

3. The method according to claim 2, characterized in that: In step 1, a Na2CO3 solution having a mass concentration of 10% is used to terminate the reaction.

4. The method according to claim 2, characterized in that: Step 1 Purification: Purification is carried out by column chromatography.

5. The method according to claim 2, characterized in that: The purification treatment in step 2 is performed by column chromatography.

6. Use of the mitochondrial-targeted viscosity-responsive AIE probe according to claim 1 in visualizing changes in cell mitochondrial viscosity.

7. The use according to claim 6, characterized in that, The application is to visualize the viscosity changes of cancer cell mitochondria by constructing the intrinsic relationship of drug action time-viscosity-fluorescence lifetime (t-η-τ).

8. A compound, characterized in that The structural formula is as follows:

9. Use of the compound according to claim 8 as a mitochondrial-targeted viscosity-responsive AIE probe.

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