Mitochondrial-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties

Abstract

The application discloses a mitochondrion-targeting near-infrared viscosity fluorescent probe with an aggregation-induced emission characteristic, and belongs to the technical field of fluorescent probes.The mitochondrion-targeting near-infrared viscosity fluorescent probe provided by the application has the aggregation-induced emission characteristic, can be used as a detection reagent to detect viscosity changes in a fatty liver, inflammation and tumor model, and has a good active oxygen generation capacity, can be used as a photosensitizer to perform a photodynamic therapy on cancer, and the detection and treatment means are simple and sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of viscosity fluorescent probes, and in particular relates to a mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced luminescence characteristics. Background Art

[0002] Viscosity, as a crucial parameter of the cellular microenvironment, plays a crucial role in material diffusion and signal transduction. Mitochondria, a crucial organelle of eukaryotic cells, are considered the primary powerhouse of the cell. The homeostasis of their microenvironment is crucial for normal life activities. Mitochondrial viscosity plays a crucial role in regulating their physiological processes. Abnormal changes in mitochondrial viscosity often lead to a variety of diseases, including inflammation, neurodegenerative diseases, and even cancer. Therefore, the development of fluorescent probes to monitor mitochondrial viscosity will provide a deeper understanding of the physiological and pathological processes associated with mitochondrial viscosity. Most existing viscosity-sensitive probes are based on the aggregation-induced quenching mechanism, whereby they emit light at low concentrations but quench at high concentrations or in aggregated states, severely limiting their effectiveness as luminescent materials. Molecules exhibiting aggregation-induced emission (AIE) properties exhibit low or very weak fluorescence when dissolved due to free intramolecular rotation. However, once aggregated or confined in a rigid environment, the restricted intramolecular motion results in intense fluorescence. Therefore, the development of highly sensitive fluorescent viscosity probes with AIE properties that can monitor mitochondrial viscosity is highly desirable.

[0003] Photodynamic therapy (PDT) has become a clinically recognized and promising cancer treatment method due to its minimally invasive, highly selective, low toxicity and side effects, and good targeted therapeutic effects. PDT activates photosensitizers (PS) under light irradiation to produce ROS, especially singlet oxygen ( 1 O2), leading to necrosis or apoptosis of cancer cells. Since ROS have the characteristics of high reactivity and short diffusion distance, the rational design of organelle-targeted PSs can significantly improve the efficacy of PDT. Considering the important role of mitochondria in cell respiration, division and apoptosis, and their high sensitivity to ROS and drug stimulation, directly targeting PSs to mitochondria can theoretically maximize the efficiency of PDT.

[130] Therefore, the development of mitochondria-targeted PSs has received extensive attention in recent years and has become an alternative to traditional nucleus-targeted drugs for killing or inhibiting cancer cells and tumorigenesis.

[0004] Currently, there are few reports on fluorescent probes with aggregation-induced emission characteristics, which can be used for visual diagnosis of various disease models (fatty liver, inflammation and even cancer), and can perform photodynamic therapy of cancer and instant evaluation of treatment effect through changes in mitochondrial viscosity. Therefore, it is necessary to develop a mitochondria-targeting near-infrared viscosity fluorescent probe with aggregation-induced emission characteristics for visual diagnosis of various disease models and photodynamic therapy of cancer, and to realize diagnosis and treatment integration. SUMMARY

[0005] In view of the defects in the prior art, the present application provides a mitochondria-targeting near-infrared viscosity fluorescent probe (POTA-OH) with aggregation-induced emission characteristics. The positive charge structure of the probe is combined with the electrostatic action of the negative membrane potential of mitochondria, so that the probe is targeted and aggregated in mitochondria. At the same time, the AIE molecule motion is limited, and the effective conjugation of the molecule is realized by limiting the free rotation of multiple rotatable phenyl groups and single bonds, thereby realizing high-sensitivity recognition of mitochondrial viscosity. At the same time, the viscosity-dependent POTA-OH can perform instant evaluation of treatment effect during the in vitro PDT process. In addition, the high-efficiency PDT of POTA-OH has also achieved success in the in vivo anti-tumor aspect, and provides an effective strategy for tumor treatment and instant evaluation of treatment effect.

[0006] To achieve the above object, the present application adopts the following technical scheme:

[0007] A mitochondria-targeting near-infrared viscosity fluorescent probe with aggregation-induced emission characteristics, the structural formula of which is:

[0008]

[0009] A preparation method of a mitochondria-targeting near-infrared viscosity fluorescent probe with aggregation-induced emission characteristics, comprising the following steps:

[0010] Step 1: 6-bromo-2,3-dihydro-1H-xanthene-4-formaldehyde, 4-methoxydiphenylamine, Pd2(dba)3, Davephos and t-BuOK are dissolved in anhydrous toluene; after heating and stirring overnight under nitrogen, cooling, removing the solvent under reduced pressure, and purifying by column chromatography, an orange solid, compound 1 (6-(4-methoxyphenyl)(phenyl)amino-2,3-dihydro-1H-xanthene-4-formaldehyde), is collected;

[0011] Step 2: 4-methylpyridine and methyl iodide are added to anhydrous acetonitrile, and the mixture is heated and stirred overnight under nitrogen. After cooling, the solvent is removed under reduced pressure, a large amount of solid is filtered off and washed with diethyl ether, and a pink solid, compound 2 (1,4-dimethylpyridine-1-iodide), is obtained;

[0012] Step 3: Compound 1, compound 2, zinc acetate and piperidine were added into ethanol, stirred under heating condition, cooled, removed the solvent under reduced pressure, and the residue was purified by column chromatography to obtain a purple-black solid, i.e. compound 3 ((E)-4-(2-(6-(4-methoxyphenyl)(phenyl)amino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-1-methylpyridine-1-iodide);

[0013] Step 4: Compound 3 and boron tribromide were added into a two-necked round-bottom flask, stirred under argon, added sodium bicarbonate, then extracted with dichloromethane, the organic layer was dried with anhydrous magnesium sulfate, the solid was filtered under reduced pressure, the solvent was removed, and the residue was purified by silica gel column chromatography to obtain a purple-black solid, i.e. POTA-OH;

[0014] The preparation reaction formula of the fluorescent viscosity probe POTA-OH described in the present application is as follows:

[0015]

[0016] The preparation process of the compound described in the present application refers to the prior art with similar structure, and the reference is J. Ouyang, L.H. Sun, Z. Zeng, C. Zeng, F. Zeng and S.Z. Wu, Angewandte Chemie International Edition, 2020, 59, 10111-10121.

[0017] The molar ratio of 6-bromo-2,3-dihydro-1H-xanthene-4-carboxaldehyde and 4-methoxydiphenylamine in step 1 is 2:5, and the temperature of the heating and stirring is 120℃;

[0018] The temperature of the heating and stirring in step 2 is 80℃;

[0019] The temperature of the stirring under heating condition in step 3 is 90℃, and the time is 10-12h;

[0020] The stirring temperature in step 4 is 0℃, and the time is 2-4h, and the silica gel column chromatography uses dichloromethane / anhydrous methanol eluent with a volume ratio of 30:1-10:1.

[0021] The fluorescent viscosity probe provided by the present application has the characteristic of aggregation-induced emission, and the near-infrared fluorescence intensity of the probe gradually increases with the increase of the environmental viscosity.

[0022] The application of a mitochondria-targeting near-infrared viscosity fluorescent probe with the characteristic of aggregation-induced emission in the preparation of a reagent for monitoring the viscosity change in mitochondria of living cells.

[0023] The application of a mitochondria-targeting near-infrared viscosity fluorescent probe with an aggregation-induced emission characteristic in the preparation of a reagent for detecting viscosity changes in an inflammation model.

[0024] The application of a mitochondria-targeting near-infrared viscosity fluorescent probe with an aggregation-induced emission characteristic in the preparation of a reagent for detecting viscosity changes in a fatty liver model.

[0025] The application of a mitochondria-targeting near-infrared viscosity fluorescent probe with an aggregation-induced emission characteristic in the preparation of a reagent for detecting viscosity changes in a tumor model.

[0026] The application of a mitochondria-targeting near-infrared viscosity fluorescent probe with an aggregation-induced emission characteristic in the preparation of a photodynamic therapy reagent.

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

[0028] (1) The fluorescence probe for detecting mitochondrial viscosity in the application uses a hydroxydiphenylamine-substituted xanthene group as an aggregation-induced fluorescence core and an electron donor (D), a pyridine cation as an electron acceptor (A), and a mitochondria-targeting group. The molecular structure has a strong push-pull electron effect and a twisted D-A effect; in a dimethyl sulfoxide / toluene mixed solvent, the fluorescence of the probe gradually increases with the increase of the toluene content, and the probe has a typical aggregation-induced fluorescence emission characteristic.

[0029] (2) The response principle of the probe to viscosity changes: due to the multiple freely rotatable phenyl and ethenyl single bonds, the molecule can freely rotate in a medium with low viscosity, and is in a twisted intramolecular charge transfer state, resulting in fluorescence quenching; when in a system with high viscosity, the free rotation is limited, the twisted intramolecular charge transfer effect is inhibited, the whole molecule is coplanar, and the probe emits strong near-infrared fluorescence.

[0030] (3) In a water-glycerol system, when the viscosity η increases from 0.89 cP to 945 cP, the near-infrared fluorescence intensity of the probe at 720 nm gradually increases, and the enhancement factor is more than 157 times. In addition, log I 720 and log η have a good linear relationship (R 2 = 0.9896), and the slope is 0.725, which has the characteristics of high sensitivity and quantitative detection of environmental viscosity.

[0031] (4) The detection of environmental viscosity by the probe has high selectivity and high sensitivity, and is not affected by other substances, polarity and pH in the biological system and environment.

[0032] (5) The probe utilizes the electrostatic interaction between the positive charge structure of the molecule and the negative membrane potential of mitochondria to target the probe to mitochondria; when applied to real-time in situ monitoring of viscosity changes in a mitochondrial viscosity cell model, it also shows high sensitivity response in vivo viscosity and inflammation mice. In addition, it is also applied to the visualization identification of fatty liver tissue sections, in vivo mouse tumors and clinical cancer patient pathological samples, with high sensitivity, reliability, visualization, fast and convenient characteristics, indicating that the probe has good application prospects in mitochondrial related biological research and medical diagnosis.

[0033] (6) The probe has good active oxygen generation capacity, which can simultaneously perform photodynamic therapy in vitro and evaluate the treatment effect, realize efficient photodynamic therapy of in vivo tumor, and has potential application value in visualization detection, disease diagnosis and treatment in biological system, and provides a very promising fluorescent reagent for realizing diagnosis and treatment integration of cancer.

[0034] (7) The detection means is simple, only including a fluorescence spectrophotometer, a laser confocal microscope and a multi-mode in vivo imaging instrument. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 For the nuclear magnetic characterization of the probe POTA-OH of the application, 1 H-NMR spectrum;

[0036] Figure 2 For the nuclear magnetic characterization of the probe POTA-OH of the application, 13 C-NMR spectrum;

[0037] Figure 3 For the nuclear magnetic characterization of the probe POTA-OH of the application, HR-MS spectrum;

[0038] Figure 4 For the fluorescence emission spectrum of the probe POTA-OH of the application in dimethyl sulfoxide-toluene mixed solvent with toluene volume content change;

[0039] Figure 5 For the curve of the relative fluorescence intensity (I / I0) of the probe POTA-OH of the application in the dimethyl sulfoxide-toluene mixed system with the volume content change;

[0040] Figure 6 For the fluorescence emission spectrum of the probe POTA-OH of the application in water-glycerol mixed solvent with glycerol volume content change;

[0041] Figure 7 For the curve of the logI of the probe POTA-OH of the application in the water / glycerol mixed system with the logη change;

[0042] Figure 8 Fluorescence emission spectra of the probe POTA-OH in different polar solvents;

[0043] Figure 9 Fluorescence emission spectra of the probe POTA-OH in PBS-glycerol (0% and 95%) mixed solvents with different glycerol volume fractions, when the pH changes from 3.0 to 10.0;

[0044] Figure 10 Selectivity column chart of the fluorescence spectra of the probe POTA-OH in glycerol in the presence of common metal ions, anions and bioactive small molecules;

[0045] Figure 11 Fluorescence co-localization imaging chart of the probe POTA-OH and commercially available mitochondria-specific dye (MTG) co-staining living cells;

[0046] Figure 12 Real-time fluorescence imaging chart of the probe POTA-OH in the viscosity change of HeLa cells stimulated by nystatin;

[0047] Figure 13 Fluorescence imaging chart of the probe POTA-OH in the viscosity change of living mice stimulated by nystatin and lipopolysaccharide, respectively;

[0048] Figure 14 Fluorescence imaging chart of the probe POTA-OH in the viscosity change of left (tumor) and right (normal) axillary tissues in a tumor mouse model;

[0049] Figure 15 Fluorescence imaging chart of the probe POTA-OH in the viscosity change of pathological tissue sections of clinical cancer patients;

[0050] Figure 16 Fluorescence imaging chart of the probe POTA-OH in the viscosity change of fatty liver tissue sections;

[0051] Figure 17 Cell survival rate chart of HeLa cells in the presence of the probe POTA-OH under dark or laser irradiation;

[0052] Figure 18 Fluorescence imaging chart of HeLa cells in the presence or absence of an indicator, 2', 7'-dichlorofluorescein diacetate (DCFH-DA), or DCFH-DA alone under laser irradiation;

[0053] Figure 19Fig. 1 is a fluorescence imaging change diagram of the fluorescent probe POTA-OH of the present application in living HeLa cells, laser-irradiated living HeLa cells and fixed HeLa cells, respectively;

[0054] Figure 20 Fig. 4 is a tumor volume change diagram of a tumor-bearing mouse treated with the fluorescent probe POTA-OH of the present application in vivo photodynamic therapy for 21 days;

[0055] Figure 21 Fig. 5 is a mouse weight change diagram of a tumor-bearing mouse treated with the fluorescent probe POTA-OH of the present application in vivo photodynamic therapy for 21 days;

[0056] Figure 22 Fig. 6 is a tumor tissue section immunohistochemical staining diagram of a tumor-bearing mouse treated with the fluorescent probe POTA-OH of the present application in vivo photodynamic therapy for 21 days. DETAILED DESCRIPTION

[0057] Example 1

[0058] Preparation and characterization of a mitochondria-targeting near-infrared viscosity fluorescent probe with aggregation-induced emission characteristics:

[0059]

[0060] (1) 6-bromo-2,3-dihydro-1H-xanthene-4-carboxaldehyde (compound 1), 289 mg, 1 mmol, 4-methoxydiphenylamine (498 mg, 2.5 mmol), Pd2(dba)3 (91.5 mg, 0.1 mmol), Davephos (40 mg, 0.1 mmol) and t-BuOK (224 mg, 2 mmol) were dissolved in 9 mL of anhydrous toluene. After stirring overnight at 120°C under nitrogen, it was cooled to 25°C, the solvent was removed under reduced pressure, and column chromatography (petroleum ether / acetone, 30:1, v / v) was used to purify and collect orange solid, which was compound 2 (123 mg, yield 19%). 1 HNMR (600 MHz, CDCl3) δ 10.19 (s, 1H), 7.31-7.26 (m, 2H), 7.13-7.04 (m, 5H), 6.95 (d, J = 8.8 Hz, 1H), 6.91-6.85 (m, 2H), 6.68-6.63 (m, 2H), 6.60 (s, 1H), 3.82 (s, 3H), 2.55 (t, J = 5.6 Hz, 2H), 2.42 (t, J = 5.6 Hz, 2H), 1.68 (q, J = 6.4 Hz, 2H).

[0061] (2) 4-Methylpyridine (Compound 3) (500 mg, 5.37 mmol) and CH3I (951 mg, 6.7 mmol) were added to 6 mL of anhydrous CH3CN. The mixture was stirred at 80°C overnight under nitrogen, then cooled to 25°C. The solvent was removed under reduced pressure, and a large amount of solid was filtered and washed with ether to obtain Compound 4 (953 mg, 70%) as a pink solid. 1 H NMR (600MHz, CDCl3) δ9.12 (d, J = 6.8 Hz, 2H), 7.85 (d, J = 6.4 Hz, 2H), 4.64 (s, 3H), 2.68 (s, 3H).

[0062] (3) Compound 2 (45 mg, 0.11 mmol), compound 4 (20 mg, 0.187 mmol), zinc acetate (52 mg, 0.286 mmol), and piperidine (51 μL) were added to 2.5 mL of ethanol. The mixture was stirred at 90°C for 12 h. After cooling to 25°C, the solvent was removed under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol, 40:1, v / v) to obtain compound 5 (33 mg, 53%) as a purple-black solid. 1 HNMR(600MHz,DMSO-d6)δ8.56(d,J=6.8Hz,2H),8.13(d,J=15.6Hz,1H),8.01(d,J=6.8Hz,2H),7.38-7.30(m,2H),7.15-6.9 5(m,8H),6.86(d,J=2.0Hz,1H),6.74(s,1H),6.60-6.51(m,2H),4.12(s,3H),3.77(s,3H),1.79-1.70(m,4H),1.24(s,2H).

[0063] (4) Compound 5 (40 mg, 0.08 mmol) and 2 mL of BBr3 were added to a two-necked round-bottom flask. After stirring at 0°C for 3 h under argon, 1 mL of NaHCO3 was added, and then extracted with DCM (3×15 mL). The organic layer was dried over anhydrous MgSO4, and the solid was filtered under reduced pressure. The solvent was removed and purified by silica gel column chromatography (dichloromethane / methanol, 30:1-10:1, v / v) to obtain a purple-black solid, POTA-OH (36 mg, yield 95%). Figure 1 As shown, 1H NMR (600MHz, DMSO-d6) δ9.52(s,1H),8.55(d,J=6.8Hz,2H),8.12(d,J=15.6Hz,1H),8.00(d,J=6.8Hz,2H),7.31(t,J=7.6Hz,2H),7.1 4-6.94(m,6H),6.85-6.78(m,3H),6.73(s,1H),6.59-6.50(m,2H),4.11(s,3H),2.57-2.53(m,2H),2.48-2.44(m,2H),1.74(m,2H). Such as Figure 2 As shown, 13 C NMR (150 MHz, DMSO-d6) δ 155.51, 153.72, 153.32, 152.97, 149.91, 147.30, 144.45, 137.93, 135.57, 130.04, 128.64, 127.67, 127.28, 125.34, 124.20, 123.66, 122.65, 118.64, 117.07, 116.64, 115.60, 111.03, 106.74, 46.60, 29.39, 24.71, 20.85. Figure 3 As shown, HR-MS m / z: [M+H] + calcd.for C 33 H 29 N2O2 + ,485.2224; measured,485.2217.

[0064] Example 2

[0065] The aggregation-induced fluorescence emission characteristics of the fluorescent probe POTA-OH in a dimethyl sulfoxide-toluene mixed solvent were studied. The fluorescent probe in Example 1 was diluted to a final concentration of 10 μmol / L with a dimethyl sulfoxide-toluene mixed solvent, and the excitation wavelength was fixed at 565 nm. The fluorescence emission spectrum of the probe was recorded as the volume content of toluene changed ( Figure 4 ), and draw the curve of the probe relative fluorescence intensity (I / I0) changing with the volume content in the dimethyl sulfoxide-toluene mixture system ( Figure 5 As the toluene volume ratio increased from 0% to 100%, the fluorescence intensity at 730 nm gradually increased and blue-shifted to 714 nm, reaching a maximum value when the toluene volume was 95%, indicating that the probe has typical aggregation-induced emission properties.

[0066] Example 3

[0067] The fluorescent probe POTA-OH in Example 1 was diluted with a mixed solvent of water and glycerol to a final concentration of 10 μmol / L, the excitation wavelength was fixed at 565 nm, and the fluorescence emission spectrum of the probe was recorded as the volume content of glycerol (or viscosity coefficient η) changed. Figure 6 ), and plot the fluorescence intensity of the probe at 720 nm (log I 720 ), the linear correlation curve of the viscosity coefficient (logη) in the water and glycerol mixture system ( Figure 7 As the glycerol volume ratio increased from 0% (0.89 cP) to 99% (945 cP), the fluorescence intensity at 720 nm gradually increased and reached a maximum value when the glycerol volume was 99%, indicating that the relative fluorescence intensity of the probe significantly increased with the increase of the environmental viscosity.

[0068] Example 4

[0069] The concentration of the fluorescent probe POTA-OH in Example 1 was kept at 10 μmol / L, and the fluorescence emission spectra of the probe in solutions of different polarities were investigated. Figure 8 As shown in the figure, the probe has a significant fluorescence enhancement in glycerol, while in other solvents of different polarity, the fluorescence intensity does not change much, indicating that the response of the probe to viscosity is basically unaffected by the polarity of the solvent.

[0070] Example 5

[0071] The concentration of the fluorescent probe POTA-OH in Example 1 was kept at 10 μmol / L, and the fluorescence spectra of the probe were investigated in PBS / glycerol systems containing 0% and 95% glycerol, respectively, as the pH changed. Figure 9 As shown in Figure 3, as the pH increased from 3.0 to 10.0, the fluorescence intensity of the probe remained basically stable, indicating that the response of the probe to viscosity was not affected by pH changes.

[0072] Example 6

[0073] The concentration of the fluorescent probe POTA-OH in Example 1 was kept at 10 μmol / L, and the selectivity of the fluorescence spectrum of the probe was investigated in the presence of common ions and bioactive small molecules. Figure 10 As shown in the figure, in a phosphate buffer solution (PBS) (2 mL, pH 7.4) system, the addition of the following substances (100 μmol / L) had almost no interference on the fluorescence intensity of the probe. Figure 10 The substances in the sample are as follows: 1. Blank; 2. 99% Glycerol; 3. Trp; 4. Cys; 5. GSH; 6. Hcy; 7. Pro; 8. Ser; 9. Ala; 10. Zn 2+ ;11.Cu 2+ ;12.Pb 2+ ;13.K+ ;14Mg 2+ ;15Na + ;16.Ba 2+ ;17.Ca 2+ ;18.Cd 2+ ; 19.Fe 2+ ; 20.Fe 3+ ;21.Br - ;22.Cl - ;23.CN - ;24.CO3 2- ; 25.H2PO4 - ; 26.HSO3 - ; 27.HSO4 - ;28.NO2 - ;29.NO3 - ;30.S 2- ;31.S2O3 2- ;32.SO4 2- .

[0074] Example 7

[0075] In order to observe whether the probe POTA-OH can target and accumulate in the mitochondria of living cells, a co-localization experiment of the probe and the commercially available mitochondria-specific dye MitoTracker Green (MTG) was performed. The adherent HeLa cells were incubated with MTG (final concentration 1.0 μmol / L) at pH 7.4 in an incubator at 37°C and 5% CO2 for 30 minutes, and then washed three times with PBS buffer (pH 7.4) to remove excess dye. The probe POTA-OH (final concentration 5 μmol / L) was then added and incubated for another 30 minutes, and the co-localization of the two was observed under a laser confocal microscope. The probe POTA-OH had a fixed excitation wavelength of 561 nm, and red fluorescence imaging was selected to collect red channel range of 650-754 nm; the fixed excitation wavelength of MTG was 488 nm, and the red channel range of 490-530 nm was collected. Figure 11 As can be seen, the fluorescent probe POTA-OH exhibits a typical red rod-shaped mitochondrial morphology and overlaps well with MTG, producing yellow overlapping fluorescence. The Pearson colocalization coefficient (PC) of the two is 0.91, indicating that the fluorescent probe POTA-OH and MTG have significant colocalization imaging and can be targeted to mitochondria.

[0076] Example 8

[0077] Adherent HeLa cells were incubated with the fluorescent probe POTA-OH (final concentration 5 μmol / L) in Example 1 in an incubator at 37°C and 5% CO2 for 30 min without washing. The fluorescence imaging of the probe was observed under a laser confocal microscope. Figure 12 As shown, the probe itself emits weak fluorescence in the cells. Then, when 5 μmol / L nystatin is added and the cells are stimulated for 30 minutes, the fluorescence emission in the cells is gradually enhanced and observed in situ. At the same time, the mitochondrial morphology changes from rod-shaped to spherical, indicating that nystatin can lead to an increase in mitochondrial viscosity and changes in mitochondrial morphology, and the probe POTA-OH can highly sensitively monitor changes in mitochondrial viscosity.

[0078] Example 9

[0079] The fluorescent probe POTA-OH (100 μL, 200 μM) in Example 1 was intraperitoneally injected into normal mice, mice treated with nystatin (viscosity mouse model), and mice treated with lipopolysaccharide (inflammation mouse model). Figure 13 As shown, very weak red fluorescence was observed in normal mice under in vivo imaging, while significant red fluorescence was observed in mice treated with nystatin and lipopolysaccharide. This indicates that intraperitoneal injection of nystatin and lipopolysaccharide can increase viscosity in mice, indicating that this probe can be used to monitor real-time changes in viscosity in live mouse models of viscosity and inflammation.

[0080] Example 10

[0081] The fluorescent probe POTA-OH (50 μL, 200 μmol / L) in Example 1 was injected into the left (tumor) and right (normal) axilla of tumor-bearing mice, and the fluorescence imaging of the probe was observed under an in vivo imaging device. Figure 14 As shown, the probe exhibits bright red fluorescence in the left axillary tumor, while the fluorescence in the right axillary normal tissue is negligible, indicating that the viscosity level in the tumor tissue is significantly higher than that in the normal tissue, and the probe POTA-OH can achieve highly sensitive visual identification of the tumor.

[0082] Example 11

[0083] The fluorescent probe POTA-OH (final concentration 20 μmol / L) in Example 1 was incubated in human benign (breast and thyroid) and malignant cancer (breast and thyroid) tissue sections, respectively. Fluorescence imaging of the probe was performed under a laser confocal microscope. Significant red fluorescence enhancement signals were observed in malignant cancer tissues, while the fluorescence signals in benign tissue sections were negligible ( Figure 15 ), indicating that the viscosity in malignant cancer tissues is significantly higher than that in benign tissues, and the probe POTA-OH has good cancer visualization and recognition capabilities.

[0084] Example 12

[0085] The fluorescent probe POTA-OH (final concentration 20 μmol / L) in Example 1 was incubated in normal liver tissue and fatty liver tissue, respectively. The fluorescence imaging of the probe was observed under a laser confocal microscope. The probe showed bright red fluorescence in fatty liver sections, while the fluorescence in normal liver tissue sections was negligible ( Figure 16 ), indicating that the viscosity level in fatty liver tissue is significantly higher than that in normal liver tissue, and the probe POTA-OH can achieve efficient visual identification of fatty liver tissue.

[0086] Example 13

[0087] The fluorescent probe POTA-OH with different concentrations (0, 0.5, 1.0, 5.0, 10.0, 15.0 μmol / L) in Example 1 was placed in the dark or under the influence of a 635 nm laser (50 mW·cm -2 , 20min) irradiation, such as Figure 17 As shown in the results, POTA-OH exhibited good biocompatibility in the dark, while after laser irradiation, it showed probe dose-dependent cytotoxicity, indicating that POTA-OH had a good photodynamic therapeutic effect on tumor cells.

[0088] Example 14

[0089] The probe POTA-OH in Example 1 was irradiated with a 635 nm laser (100 mW·cm -2 Real-time in situ fluorescence imaging of HeLa cells under irradiation with or without the indicator 2'7'-dichlorofluorescein diacetate (DCFH-DA) or with DCFH-DA alone. Figure 18 As shown, when the probe and DCFH-DA co-existed, the intracellular fluorescence intensity was significantly enhanced, while when DCFH-DA or the probe existed alone, the intracellular fluorescence did not change significantly, indicating that the probe effectively produced reactive oxygen species in the cells.

[0090] Example 15

[0091] The probe POTA-OH in Example 1 was exposed to living HeLa cells and a 635 nm laser (100 mW·cm -2 ) irradiated live HeLa cells and fixed HeLa cells (fixed with polychloroformaldehyde to shrink the cells, resulting in increased viscosity of the fixed cells) were co-incubated separately. Figure 19As shown, with the increase of laser irradiation time, the probe produces a photodynamic therapeutic effect in living cells, inducing gradual cell apoptosis, resulting in a significant increase in intracellular viscosity and an increase in fluorescence intensity, which is consistent with the fluorescence phenomenon in fixed cells. This further proves that the probe has good in vitro cancer cell photodynamic therapy ability and can achieve instant evaluation of the treatment effect through changes in cell viscosity.

[0092] Example 16

[0093] The probe POTA-OH (3 mM, 100 μL / 200 mm 3 Tumor) was injected into the mouse tumor by intratumoral injection for 2 h, and then the tumor was illuminated by 635 nm laser (100 mW·cm -2 The tumor site was irradiated with laser for 30 min and the weight and tumor volume of the mice were measured every 3 days. The mice injected with PBS were then irradiated under the same conditions (PBS + Laser) and the mice injected with POTA-OH but not irradiated were used as parallel control groups. Figure 20 As shown in the figure, the average tumor volume of the PBS+Laser group and the POTA-OH group increased rapidly, only the POTA-OH+Laser (635nm, 100mW·cm -2 ) experimental group, the tumor volume gradually decreased, indicating that the tumor was inhibited, further confirming that POTA-OH exhibited excellent PDT effect in vivo. Figure 21 As shown in the figure, the weight of mice in the control and experimental groups remained almost unchanged, indicating that the probe had almost no side effects on photodynamic therapy of tumors. More importantly, after 21 days, tumor tissues were dissected from all experimental mice and immunohistochemically stained with Ki67. Compared with the control group, the cancer cells in the POTA-OH+Laser group almost completely lost their ability to proliferate ( Figure 22 ), further confirming the therapeutic effect of POTA-OH, which can effectively inhibit tumor growth.

Claims

1. A mitochondrial-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission characteristics, characterized in that: The structural formula is: 。 2. Use of a mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties as claimed in claim 1 in the preparation of a reagent for monitoring viscosity changes in mitochondria of living cells.

3. Use of a mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties as described in claim 1 in the preparation of a reagent for detecting viscosity changes in an inflammatory model.

4. Use of a mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties as claimed in claim 1 in the preparation of a reagent for detecting viscosity changes in a fatty liver model.

5. Use of a mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties as claimed in claim 1 in the preparation of a reagent for detecting viscosity changes in tumor models.

6. Use of the mitochondria-targeted near-infrared viscosity fluorescent probe with aggregation-induced emission properties as claimed in claim 1 in the preparation of photodynamic therapy reagents.

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