Targeting Golgi apparatus AIE ionic probe, and preparation method and application thereof

Abstract

The invention provides an aggregation-induced emission (AIE) ionic probe (E)-4-(2-(5-(4-(diphenylamino) phenyl) thiophene-2-yl) vinyl)-1-(4-sulfanilamide acyl benzyl) quinoline-1-onium hexafluorophosphoric acid (V) salt of a targeted golf apparatus, namely a fluorescent probe TTQ-GA, a preparation method of the fluorescent probe TTQ-GA and application of the fluorescent probe TTQ-GA in the aspect of detection. The benzenesulfonamide group of the probe can be selectively combined with a Golgi apparatus protein cyclooxygenase 2 (COX-2) inhibitor SC-558 to realize Golgi apparatus targeting; due to the hydrophobic triphenylamine part and the hydrophilic quinoline cation part of the probe, the probe has amphipathy; the probe does not emit fluorescence in an aqueous solution, and can emit strong fluorescence when molecular movement is limited and excited, so that no-wash marking of the Golgi apparatus is realized; the probe generates a large amount of ROS under light excitation, promotes local lipid peroxidation increase of Golgi apparatus, and promotes cancer cell apoptosis.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent probe molecules, specifically to an ionic probe that exhibits Golgi-targeted aggregation-induced emission, its preparation method, and its applications. Background Technology

[0002] The Golgi apparatus, a core secretory organelle in eukaryotic cells, is responsible for post-translational modification of proteins, sorting and transport, regulation of lipid synthesis, and lysosome formation. Its unique polar membrane structure plays a crucial regulatory role in cellular homeostasis, and Golgi structural abnormalities or functional disorders have been proven to be closely related to a variety of major diseases, including neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), tumor metastasis, and metabolic syndrome. Because the Golgi apparatus is highly dependent on the redox balance of the membrane system, its multilayered membrane structure is abnormally sensitive to oxidative stress factors such as reactive oxygen species (ROS). Therefore, precise dynamic tracing and spatiotemporally resolved imaging techniques targeting the Golgi apparatus are of great significance for elucidating its pathological mechanisms and developing targeted therapeutic strategies.

[0003] In the development of Golgi-specific fluorescent probes, existing probes generally face problems such as poor membrane system permeability and insufficient polar retention capacity, resulting in significant limitations in long-term in vivo imaging. The luminescence mechanism of traditional probes based on planar conjugated structures is prone to aggregation fluorescence quenching (ACQ) in the dense Golgi membrane structure, severely affecting imaging resolution and stability. In recent years, probes with aggregation-induced emission (AIE) properties have shown unique advantages in maintaining dynamic tracking of the Golgi membrane system due to their anomalous "hydrophobic aggregation-enhanced luminescence" characteristic. AIE probes can not only achieve long-term imaging with ultra-high signal-to-noise ratios, but also simultaneously conduct drug delivery and efficacy monitoring through functionalization modifications.

[0004] In recent years, significant progress has been made in the development of photoactivated aggregation-induced emission materials, but their synthesis routes still face technological bottlenecks such as lengthy multi-step organic reactions and difficulties in targeted functionalization modification. Existing systems generally suffer from two major technical defects: first, the photophysical response mode is singular, making it difficult to simultaneously achieve multi-dimensional in-situ monitoring of subcellular organelle microenvironment parameters (such as pH, viscosity, and reactive oxygen species gradients); second, diagnostic and therapeutic functions are disconnected, with a lack of molecular-level synergistic design between the luminescent unit and the therapeutic module. More importantly, current oxidative stress research largely relies on exogenous stimuli (such as the exogenous introduction of H2O2, doxorubicin, or peroxisome activators). This intervention method easily induces an imbalance in the intracellular redox network, causing subcellular organelle-specific stress signals to be masked by system noise, severely restricting the accurate analysis of dynamic changes in the microenvironment of key organelles such as the Golgi apparatus. Summary of the Invention

[0005] Objective of the Invention: To address the shortcomings and defects of existing technologies, this invention provides an ionic probe for Golgi apparatus-targeted aggregation-induced emission, its preparation method, and its applications. The benzenesulfonamide group of this probe selectively binds to the Golgi apparatus protein cyclooxygenase 2 (COX-2) inhibitor SC-558, achieving Golgi apparatus targeting. The probe's hydrophobic triphenylamine moiety and hydrophilic quinoline cation moiety give it amphiphilic properties. The probe does not fluoresce in aqueous solution but emits strong fluorescence when molecular motion is restricted and excited, enabling wash-free labeling of the Golgi apparatus. Under photoexcitation, the probe generates a large amount of ROS, promoting increased local lipid peroxidation in the Golgi apparatus and promoting cancer cell apoptosis.

[0006] Technical solution: The present invention provides an ionic probe for Golgi apparatus-targeted aggregation-induced emission, characterized in that: the chemical structural formula of the ionic near-infrared fluorescent probe is as follows: .

[0007] The probe's specific molecular structure, specifically the benzenesulfonamide group, selectively binds to the Golgi protein cyclooxygenase 2 (COX-2) inhibitor SC-558, achieving Golgi targeting. The fluorescent probe molecule TTQ-GA, possessing AIE properties, is amphiphilic (a hydrophobic triphenylamine moiety and a hydrophilic quinoline cation moiety). Due to its non-radiative transition, it does not fluoresce in aqueous solution, but emits strong fluorescence when molecular motion is restricted and excited, thus achieving wash-free labeling of the Golgi apparatus. Furthermore, the fluorescent probe molecule generates a large amount of ROS under photoexcitation, promoting increased local lipid peroxidation in the Golgi apparatus, thereby effectively promoting cancer cell apoptosis.

[0008] The present invention discloses a method for preparing an ionic probe for Golgi-targeted aggregation-induced emission, characterized in that: 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde (TTC), piperidine, and 4-methyl-1-(4-sulfonamidobenzyl)quinoline-1-onium hexafluorophosphate (V) (Q-SBN) are placed in a reaction flask, ethanol (as a solvent in the reaction system) is added to the reaction flask, the mixture is heated to not less than 85°C under nitrogen protection, the reaction solution is cooled to room temperature, the organic phases are extracted and combined, dried and concentrated under reduced pressure to obtain a crude product, and the crude product is purified by column chromatography using dichloromethane / methanol as the eluent to obtain the fluorescent probe (E)-4-(2-(5-(4-(diphenylamino)phenyl)thiophene-2-yl)vinyl)-1-(4-sulfamylbenzyl)quinoline-1-onium hexafluorophosphate (V), i.e., the fluorescent probe TTQ-GA.

[0009] The molar ratio of 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde (TTC) to 4-methyl-1-(4-sulfonamidobenzyl)quinoline-1-onium hexafluorophosphate (V) (Q-SBN) is 1:1 to 1:10.

[0010] The reaction time is 4h to 48h.

[0011] The dichloromethane / methanol used as an eluent has a feed ratio of 1:1 to 100:1.

[0012] The present invention relates to the application of a Golgi AIE ion-type probe in detection.

[0013] For in vitro cells, the fluorescent probe was dissolved in DMEM culture medium at a concentration of 10 μM. HepG2, HeLa, or A549 live cells were incubated in a medium containing the fluorescent probe, and after 30 min of illumination, the amount of ROS generated by the fluorescent probe under photoexcitation was measured as an evaluation index of TTQ-GA phototoxicity. The detection process for the fluorescent probe's localization to the Golgi apparatus in cells was as follows: the fluorescent probe was dissolved in DMEM culture medium at a concentration of 10 μM; HepG2, A549, or HeLa live cells were co-incubated in a culture medium containing both the fluorescent probe and a commercially available Golgi-Trackergreen localization probe for 30 min, and the co-localization effect of the two was observed under confocal microscopy as an evaluation index of the fluorescent probe's localization to the Golgi apparatus.

[0014] The process of resisting photobleaching by the fluorescent probe localizing to the Golgi apparatus in cells is as follows: the fluorescent probe is dissolved in DMEM culture medium at a concentration of 10 μM; HepG2 cells are incubated with culture medium containing the fluorescent probe for 30 min; and the degree of fluorescence attenuation of the fluorescent probe in live cells under confocal laser scanning is measured as an evaluation index for the resistance to photobleaching by the fluorescent probe staining.

[0015] The detection process of ROS generation in cells by the fluorescent probe is as follows: prepare a culture medium containing fluorescent probe (10 μM) and DCFH-DA solution (10 μM), incubate HepG2 live cells in the culture medium containing fluorescent probe for 30 min, and use the degree of increase of green fluorescence under confocal laser scanning as the evaluation index for ROS generation in cells by the fluorescent probe.

[0016] This fluorescent probe, when used to stain the Golgi apparatus, eliminates the need for rinsing off excess dye with a buffer solution after staining, allowing for direct subsequent imaging.

[0017] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The fluorescent probe of the present invention modifies its molecular structure to achieve specific targeting of the Golgi apparatus in living cells, thus exhibiting high selectivity. The fluorescent group in the fluorescent probe of the present invention emits fluorescence in the near-infrared region, thus avoiding interference from the autoluminescence of organisms during fluorescence detection. The fluorescent probe molecules of the present invention possess AIE properties and are amphiphilic. Due to non-radiative transitions, they do not emit fluorescence in aqueous solution, but emit strong fluorescence when molecular motion is restricted and excited, thereby achieving wash-free labeling of the Golgi apparatus. The fluorescent probe of the present invention has good biocompatibility; after incubating cells in a medium containing TTQ-GA (100 μM) for 24 hours, the cell viability is still greater than 85%. The fluorescent probe of the present invention exhibits excellent phototoxicity; after incubation in a medium containing TTQ-GA (5 μM) and illumination for 30 minutes, followed by culturing cells in an incubator for 24 hours, the cell viability is less than 50%. The fluorescent probe of this invention can generate a large amount of ROS under photoexcitation, which promotes the increase of local lipid peroxidation in the Golgi apparatus, and thus can effectively promote apoptosis of cancer cells. Attached Figure Description

[0018] Figure 1 This is a synthetic route diagram of the fluorescent probe of the present invention; Figure 2 This is the UV absorption spectrum of the fluorescent probe of the present invention in dimethyl sulfoxide; Figure 3 The emission spectra of the maximum fluorescence intensity of the fluorescent probe of the present invention in mixed solvents of different ratios of dimethyl sulfoxide / toluene are shown. Figure 4 The spectrum shows the maximum fluorescence intensity ratio of the fluorescent probe of the present invention in mixed solvents of different proportions of dimethyl sulfoxide / toluene; Figure 5 The emission spectrum of the fluorescent probe of the present invention in toluene or aqueous solvent is the maximum fluorescence intensity. Figure 6 This is a confocal fluorescence imaging image of the fluorescent probe of the present invention and the commercial Golgi-Tracker Green probe in HepG2 cells after co-incubation; A in the figure is the co-localization map between TTQ-GA and Golgi-Tracker Green; B is a magnified combined image of TTQ-GA and Golgi-Tracker Green; C is the overlap coefficient and Pearson co-localization coefficient map between TTQ-GA and Golgi-Tracker Green. Figure 7 This is a time-dependent fluorescence imaging diagram of the fluorescent probe of the present invention in HepG2 cells; Figure 8This is a fluorescence imaging image of ROS generated in HepG2 cells by the fluorescent probe of the present invention. Figure 9 The image shows the dark cytotoxicity of the fluorescent probe of the present invention to HepG2, HeLa and A549 cells. Figure 10 This is a diagram showing the phototoxicity of the fluorescent probe of the present invention to HepG2, HeLa, and A549 cells. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0020] The fluorescent probe of this invention, (E)-4-(2-(5-(4-(diphenylamino)phenyl)thiophene-2-yl)vinyl)-1-(4-sulfamethoxybenzyl)quinoline-1-onium hexafluorophosphate (V), namely the fluorescent probe TTQ-GA, is prepared by the following method, the synthesis method of which is attached. Figure 1 As shown.

[0021] This compound uses hydrophobic triphenylamine as a donor, thiophene as a π-bridge, hydrophilic quinoline cation as an acceptor, and benzenesulfonamide as a Golgi targeting group to construct an aggregation-induced emission fluorescent probe. The synthesis route is simple, with high yield and low cost. It has excellent resistance to photobleaching and emits in the near-infrared region, effectively resisting background interference during fluorescence detection and enabling wash-free imaging. The benzenesulfonamide group of this probe selectively binds to the Golgi protein cyclooxygenase 2 (COX-2) inhibitor SC-558, achieving Golgi targeting. In addition, the fluorescent probe of this invention has a strong D-π-A effect, which can generate a large amount of ROS under photoexcitation, promoting local lipid peroxidation in the Golgi apparatus and thus effectively promoting cancer cell apoptosis.

[0022] Specific steps: 1 mmol of TTC (TTC and Q-SBN in this invention are prepared using the synthetic method disclosed in the following literature: Sensors & Actuators: B. Chemical 358 (2022) 131471), 200 μL of piperidine, and 2 mmol of Q-SBN were placed in a 25 mL two-necked flask. 20 mL of anhydrous ethanol was then added to the flask. Under nitrogen protection, the reaction mixture was heated to 85°C and stirred for 8 hours. After cooling the reaction solution to room temperature, the organic phases were extracted and combined, then dried and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using dichloromethane / methanol as the eluent (eluent: dichloromethane:methanol = 7:1, v:v). A dark purple solid was obtained (yield 73.5%). 1H NMR (600MHz, DMSO) δ 9.61 (s, 1H), 8.57 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 8.02 (s,1H), 7.81 (dd, J = 6.4, 2.0 Hz, 4H), 7.50 (dd, J = 8.7, 2.0 Hz, 4H), 7.29 (d, J = 7.5 Hz, 7H), 7.09 – 6.91 (m, 10H), 6.41 (s, 2H). 13 C NMR (151 MHz, DMSO) δ146.81 (d, J = 5.8 Hz), 129.90 – 128.83 (m), 127.66 – 126.89 (m), 126.63 –126.27 (m), 126.20 (s), 124.56 – 123.86 (m), 123.47 (d, J = 39.0 Hz), 123.12(s), 39.52 (s). HR-MS: m / z calcd for C 40 H 32 N3O2S2 650.19305; found, 650.20148[M] + .

[0023] A 10 μM solution of the fluorescent probe TTQ-GA in dimethyl sulfoxide was prepared, and the results were measured using a UV spectrophotometer. The results are shown in the attached figure. Figure 2 .like Figure 2 It can be seen that the maximum absorption peak of the fluorescent probe molecule TTQ-GA prepared in this invention is around 585 nm.

[0024] 10 μM fluorescent probe TTQ-GA was prepared in mixed solutions of dimethyl sulfoxide / toluene at different ratios, and the results were measured using a fluorescence-visible spectrophotometer. The results are shown in the attached figure. Figure 3 .from Figure 3 It can be seen that the fluorescence emission intensity in the TTQ-GA mixed solution is significantly enhanced with the increase of the proportion of poor solvent. Meanwhile, the maximum emission peaks in the toluene solvent all fall in the near-infrared I region, beyond 700 nm, exhibiting significant near-infrared fluorescence emission performance.

[0025] The maximum fluorescence intensity ratio (I / I0) of the 10 μM fluorescent probe TTQ-GA in mixed solutions of different proportions of dimethyl sulfoxide / toluene is shown in the attached figure. Figure 4.from Figure 4 It can be seen that the fluorescent probe TTQ-GA has obvious AIE performance, that is, the fluorescence intensity gradually increases with the increase of molecular aggregation degree.

[0026] A comparison of the fluorescence emission intensity of the 10 μM fluorescent probe TTQ-GA in aqueous and toluene solutions. Figure 5 It is known that the fluorescent probe TTQ-GA of the present invention has a higher contrast in response in the aggregated state compared with that in aqueous solution. The fact that TTQ-GA has almost no fluorescence emission in aqueous solution also verifies that TTQ-GA has the ability to perform wash-free imaging.

[0027] Confocal fluorescence imaging of 10 μM fluorescent probe TTQ-GA co-incubated with a commercial Golgi probe (Golgi-tracker Green) in HepG2 cells. Figure 6 It can be seen that the Pearson coefficient for co-localization of the fluorescent probe TTQ-GA with commercial fluorescent probes is 0.982, indicating that the fluorescent probe TTQ-GA has a very high Golgi localization ability.

[0028] A time-dependent fluorescence imaging experiment was conducted in HepG2 cells to prepare a 10 μM fluorescent probe, TTQ-GA. HepG2 cells were seeded into confocal imaging cell culture dishes, and 1 mL of the appropriate culture medium (containing 10% fetal bovine serum) was added. The cells were incubated in a constant temperature incubator (5% CO2, 37℃) for 24 hours. The cells were then washed three times with 1 mL of PBS, and then incubated again with culture medium containing 10 μM TTQ-GA. Finally, the changes in intracellular fluorescence emission intensity at different time points were observed under a confocal microscope. The excitation wavelength was 585 nm, and the fluorescence acquisition range was 650-750 nm. Figure 7 It is known that the fluorescent probe TTQ-GA of the present invention has excellent photostability in HepG2 cells.

[0029] A time-dependent ROS generation experiment was conducted in HepG2 cells using a 10 μM fluorescent probe, TTQ-GA. HepG2 cells were seeded into confocal imaging cell culture dishes, and 1 mL of the appropriate culture medium (containing 10% fetal bovine serum) was added. The cells were incubated in a constant temperature incubator (5% CO2, 37℃) for 24 hours. The cells were then washed three times with 1 mL of PBS, and then incubated with culture medium containing 10 μM TTQ-GA and 10 μM DCFH-DA (which can be oxidized by ROS to 2,7-dichlorodihydrofluorescein, emitting bright green light, used to assess the generation of total reactive oxygen species (ROS)). Finally, the changes in intracellular fluorescence emission intensity at different time points were observed under a confocal microscope. The excitation wavelength was 488 nm, and the fluorescence acquisition range was 500-600 nm. Figure 8It is known that the fluorescent probe TTQ-GA of the present invention has the ability to generate a large amount of ROS in cells under photoexcitation.

[0030] Cellular toxicity assay of the fluorescent probe TTQ-GA. Cell viability was determined by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). HepG2 cells (human liver cancer cells), HeLa cells (human cervical cancer cells), and A549 cells (human lung cells) were seeded in 96-well plates, and 100 μL of medium containing 10% fetal bovine serum (FBS) was added. The cells were incubated at 37°C for 24 hours in 5% CO2. The medium for HepG2, HeLa, and A549 cells was then replaced with 100 μL of medium containing different concentrations of TTQ-GA (0, 0.5, 1, 5, 10, 20, 40 μM) and incubated for another 24 hours. The medium was then removed, and the cells were washed with 100 μL of PBS. 100 μL of medium containing 0.05% MTT was added to each well, and the cells were incubated for another 4 hours. After culture, the culture medium was removed, and 150 μL of dimethyl sulfoxide was added to each well. The mixture was then shaken slowly for 10 minutes to dissolve the formazan formed in the wells. Finally, the absorbance of each well at 490 nm was measured using a microplate reader and compared with the absorbance of the control group (100% viability) to obtain the relative cell viability at different concentrations of the fluorescent probe. Figure 9 It is evident that the fluorescent probe of this invention has good biocompatibility.

[0031] Phototoxicity assay of the fluorescent probe TTQ-GA. Cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. HepG2 cells (human liver cancer cells), HeLa cells (human cervical cancer cells), and A549 cells (human lung cells) were seeded in 96-well plates, and 100 μL of medium containing 10% fetal bovine serum (FBS) was added. The cells were incubated at 37°C for 24 hours in 5% CO2. The medium for HepG2, HeLa, and A549 cells was then replaced with 100 μL of medium containing different concentrations of TTQ-GA (0, 0.5, 1, 5, 10, 20, 40 μM), and the concentration was adjusted to 10 mW·cm⁻¹. -2Cells were exposed to white light for 30 minutes and cultured for another 24 hours. The culture medium was removed, and the cells were washed with PBS (100 μL). 100 μL of medium containing 0.05% MTT was added to each well, and the cells were cultured for another 4 hours. After culture, the culture medium was removed, and 150 μL of dimethyl sulfoxide was added to each well. The cells were then shaken slowly for 10 minutes to dissolve the formazan formed in the wells. Finally, the absorbance of each well at 490 nm was measured using a microplate reader and compared with the absorbance of the control group (100% viability) to obtain the relative cell viability at different concentrations of the fluorescent probe. Figure 10 It is evident that the fluorescent probe of this invention possesses excellent photodynamic ablation capabilities for cancer cells.

Claims

1. An ionic probe for Golgi-targeted aggregation-induced emission (AIE), characterized in that: The chemical structural formula of this ionic near-infrared fluorescent probe is: 。 2. The method for preparing the ionic probe for Golgi-targeted aggregation-induced emission according to claim 1, characterized in that: 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde (TTC), piperidine, and 4-methyl-1-(4-sulfonamidobenzyl)quinoline-1-onium hexafluorophosphate (V) (Q-SBN) were placed in a reaction flask. Ethanol was added to the flask, and the mixture was heated to at least 85°C under nitrogen protection. The reaction solution was cooled to room temperature, the organic phases were extracted and combined, dried, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using dichloromethane / methanol as the eluent to obtain the fluorescent probe (E)-4-(2-(5-(4-(diphenylamino)phenyl)thiophene-2-yl)vinyl)-1-(4-sulfamylbenzyl)quinoline-1-onium hexafluorophosphate (V), i.e., the fluorescent probe TTQ-GA.

3. The method for preparing the ionic probe for Golgi-targeted aggregation-induced emission according to claim 4, characterized in that: The molar ratio of 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde (TTC) to 4-methyl-1-(4-sulfonamidobenzyl)quinoline-1-onium hexafluorophosphate (V) (Q-SBN) is 1:1 to 1:

10.

4. The method for preparing the ionic probe for Golgi-targeted aggregation-induced emission according to claim 4, characterized in that: The reaction time is 4h to 48h.

5. The method for preparing the ionic probe for Golgi-targeted aggregation-induced emission according to claim 4, characterized in that: The dichloromethane / methanol used as an eluent has a feed ratio of 1:1 to 100:1.

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