Acridine orange-polythiophene fluorescent probe and synthesis method and application thereof

Abstract

The application discloses a synthesis method and application of an acridine orange-polythiophene fluorescent probe, and the structural formula of the fluorescent probe is shown in the following formula: wherein m is an integer of 5-30, and n=100-m.The main chain of the fluorescent probe is a cationic polythiophene, which can enter a cell nucleus and be combined with nucleic acid through electrostatic action; the side chain modified acridine orange enhances the fluorescence intensity of the polymer and is combined with nucleic acid by embedding; and the side chain modified quaternary ammonium salt can reduce cytotoxicity.The fluorescent probe has the advantages of good distinguishability, good chemical stability, small sample consumption and the like, and is a novel fluorescent probe for high-selectivity recognition and inhibition of ecDNA replication in cancer cells in a physiological system.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid fluorescence detection technology, specifically relating to an acridine orange-polythiophene fluorescent probe for recognizing and inhibiting ecDNA replication in cancer cells and its synthesis method. Background Technology

[0002] The cure rate for cancer remains unsatisfactory, primarily due to tumor heterogeneity. During tumor growth, through multiple divisions and proliferations, daughter cells exhibit molecular or genetic changes, leading to variations in growth rate, invasiveness, and drug resistance. One factor influencing tumor heterogeneity is extrachromosomal DNA (ecDNA). ecDNA is a double-stranded circular structure, ranging in size from 1 to 3 Mb. While almost never detected in normal cells, ecDNA has been found in 40% of cancer types and nearly 90% of xenograft models of brain tumors from patients. Studies have revealed that almost all oncogenes can amplify on ecDNA or on ecDNA and chromosomes, not on normal chromosomes. This suggests that these genes may have significant mobility, leading to greater tumor diversity and heterogeneity, allowing oncogenes to rapidly reach and maintain high levels. Therefore, developing methods to identify and inhibit the replication of ecDNA within tumors is both necessary and urgent.

[0003] Polythiophenes not only possess the common characteristics of conjugated polymers, but also have their own unique advantages. Polythiophenes can enter the cell nucleus and bind to nucleic acids through electrostatic interactions. Acridine orange is a classic nucleic acid dye that can bind to double-stranded DNA through intercalation, or to single-stranded RNA through ion interactions and dye stacking. Summary of the Invention

[0004] The purpose of this invention is to provide an acridine orange-polythiophene fluorescent probe with advantages such as good distinguishability, good chemical stability, and low sample consumption, and to provide a synthesis method and application for this fluorescent probe.

[0005] To achieve the above objectives, the structural formula of the acridine orange-polythiophene fluorescent probe used in this invention is as follows:

[0006]

[0007] In the formula, m is an integer from 5 to 30, and n = 100 - m.

[0008] The synthesis method of the above-mentioned acridine orange-polythiophene fluorescent probe includes the following steps:

[0009] 1. Synthesis of thiophene-acridine orange derivatives

[0010] Acridine orange base was dissolved in toluene, heated to 70–80 °C, stirred for 30–35 min, then 3-(3-bromo)butoxy-4-methylthiophene was added, heated to 105–110 °C, refluxed under argon atmosphere for 22–24 h, washed with toluene, separated and purified to obtain the thiophene-acidine orange derivative with the following structural formula;

[0011]

[0012] 2. Synthesis of thiophene quaternary ammonium salt derivatives

[0013] 3-(3-bromo)butoxy-4-methylthiophene and triethylamine were added to tetrahydrofuran, heated to 70-75 °C, refluxed for 46-48 h, and separated and purified to obtain a thiophene quaternary ammonium salt derivative with the following structural formula;

[0014]

[0015] 3. Synthesis of acridine orange-polythiophene fluorescent probe

[0016] Thiophene-acridine orange derivative and thiophene quaternary ammonium salt derivative were dissolved in chloroform. The resulting solution was then added dropwise to a chloroform solution of ferric chloride. The mixture was stirred at room temperature for 46–48 h under a nitrogen atmosphere. The solvent was evaporated to dryness, and the resulting solid was dissolved in methanol. Then, hydrazine hydrate was added for reduction, and the mixture was filtered. The filtrate was collected, and methanol was removed by rotary evaporation. The resulting solid was dissolved in dimethyl sulfoxide and water, and purified by dialysis to obtain the acridine orange-polythiophene fluorescent probe.

[0017] In step 1 above, the preferred molar ratio of acridine orange to 3-(3-bromo)butoxy-4-methylthiophene is 1:0.5 to 0.8.

[0018] In step 2 above, the preferred molar ratio of triethylamine to 3-(3-bromo)butoxy-4-methylthiophene is 1:26 to 28.

[0019] In step 3 above, the preferred molar ratio of thiophene-acridine orange derivative to thiophene quaternary ammonium salt derivative is m:n, where m is an integer from 5 to 30 and n = 100-m.

[0020] The fluorescent probe of this invention can be used to prepare anti-tumor recognition probes targeting ecDNA.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] 1. This invention uses cationic polythiophene as the main chain, introduces acridine orange, a group that can specifically bind to nucleic acids, and a hydrophilic quaternary ammonium salt to prepare an acridine orange-polythiophene fluorescent probe. The main chain of this fluorescent probe can enter the cell nucleus and bind to nucleic acids through electrostatic interaction. The acridine orange modified with side chains enhances the fluorescence intensity of the polymer and embeds itself into nucleic acids for binding, while the quaternary ammonium salt modified with side chains can reduce cytotoxicity.

[0023] 2. This invention utilizes acridine orange and polythiophene to bind with low molecular weight nucleic acids, thereby inhibiting tumor cell proliferation by suppressing the replication of ecDNA in tumor cells. It has the advantages of high recognition specificity, good biocompatibility, good chemical stability, and low sample consumption. It is a novel fluorescent probe that can highly selectively recognize and inhibit the replication of ecDNA in cancer cells in physiological systems. Attached Figure Description

[0024] Figure 1 This is the 1H NMR spectrum of the acridine orange-polythiophene fluorescent probe P(TBA5-co-TBT95) synthesized in Example 1.

[0025] Figure 2 This is the 1H NMR spectrum of the acridine orange-polythiophene fluorescent probe P (TBA10-co-TBT90) synthesized in Example 2.

[0026] Figure 3 This is the 1H NMR spectrum of the fluorescent probe PTBT synthesized in Comparative Example 1.

[0027] Figure 4 This is a fluorescence confocal image of the acridine orange-polythiophene fluorescent probe P (TBA5-co-TBT95) synthesized in Example 1 after co-incubation with three cell lines.

[0028] Figure 5 This is a fluorescence confocal image of the acridine orange-polythiophene fluorescent probe P (TBA10-co-TBT90) synthesized in Example 2 after co-incubation with three cell lines.

[0029] Figure 6 This is a fluorescence confocal image of the fluorescent probe PTBT synthesized in Comparative Example 1 after co-incubation with three cell lines.

[0030] Figure 7 The images are flow cytometry results of the fluorescent probes synthesized in Examples 1 and 2 and Comparative Example 1 after co-incubation with COLO320DM cells.

[0031] Figure 8 These are flow cytometry results of the fluorescent probes synthesized in Examples 1 and 2 and Comparative Example 1 after co-incubation with PC3 cells.

[0032] Figure 9The images are flow cytometry results of HCT116 cells after co-incubation of the fluorescent probes synthesized in Examples 1 and 2 and Comparative Example 1.

[0033] Figure 10 This is a graph showing the cytotoxicity results of the fluorescent probe PTBT synthesized in Comparative Example 1 in three cell lines.

[0034] Figure 11 The figure shows the cytotoxicity test results of the acridine orange-polythiophene fluorescent probe P(TBA5-co-TBT95) synthesized in Example 1 in three cell lines.

[0035] Figure 12 This is a cytotoxicity assay of the acridine orange-polythiophene fluorescent probe P (TBA10-co-TBT90) synthesized in Example 2 in three cell lines.

[0036] Figure 13 The image shows the transwell migration and invasion results of HCT116 cells after treatment with the acridine orange-polythiophene fluorescent probe P (TBA5-co-TBT95) synthesized in Example 1.

[0037] Figure 14 The image shows the results of transwell migration and invasion experiments of PC3 cells after treatment with the acridine orange-polythiophene fluorescent probe P (TBA5-co-TBT95) synthesized in Example 1.

[0038] Figure 15 This is a diagram showing the scratch assay results of PC3 cells treated with the acridine orange-polythiophene fluorescent probe P(TBA5-co-TBT95) synthesized in Example 1. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments. Example 1

[0040] 1. Synthesis of thiophene-acridine orange derivatives

[0041] 4 g of acridine orange was dissolved in 100 mL of 4 mol / L sodium hydroxide aqueous solution. After 30 min, the acridine orange alkaloid was extracted with dichloromethane, washed with water, dried over sodium sulfate, and the solvent was removed by rotary evaporation to obtain dried acridine orange alkaloid. 8 mL of toluene and 0.16 g (0.60 mmol) of acridine orange alkaloid were added to a dry round-bottom flask, heated to 80 °C, and stirred for 30 min. Then, 0.1 g (0.40 mmol) of 3-(3-bromo)butoxy-4-methylthiophene was added, and the mixture was heated to 110 °C and refluxed under an argon atmosphere for 24 h. The reactants were washed with toluene, and the resulting solid was dissolved in methanol. The crude product was purified by thin-layer chromatography to obtain the thiophene-acidine orange derivative (TBA). The reaction equation is as follows:

[0042]

[0043] The structural characterization data of the obtained product are as follows: 1 H NMR (600 MHz, Chloroform- d δ: 8.63 (s, 1H), 7.89 (d, J = 9.2 Hz, 2H), 7.54 (d, J = 2.4 Hz, 2H), 7.03 (dd, J = 9.3, 2.5 Hz, 2H), 6.81 (dd, J = 3.1, 1.3 Hz, 1H), 6.25 (d, J = 3.3 Hz, 1H), 5.09(t, J = 8.3 Hz, 2H), 4.19 (d, J = 5.5 Hz, 2H), 3.24 (s, 11H), 2.30 (d, J =6.3 Hz, 2H), 2.23 (s, 2H), 2.02 (d, J = 1.1 Hz, 3H); LC-MS: [M+H] + The theoretical value of m / z is 434.2261, and the measured value is 434.2263.

[0044] 2. Synthesis of thiophene quaternary ammonium salt derivatives

[0045] 60 mL of dehydrated tetrahydrofuran was added to a round-bottom flask, followed by the sequential addition of 0.53 g (2.12 mmol) of 3-(3-bromo)butoxy-4-methylthiophene and 7.4 mL (52.90 mmol) of triethylamine. The mixture was heated to 72 °C and refluxed for 48 h. The tetrahydrofuran was removed by rotary evaporation to obtain the thiophene quaternary ammonium salt derivative (TBT). The reaction equation is as follows:

[0046]

[0047] The structural characterization data of the obtained product are as follows: 1 H NMR (600 MHz, Chloroform-d) δ: 6.83 (d,1H), 6.14 (d, 1H), 3.98 (t, 2H), 3.60-3.35 (m, 8H), 2.32-2.07 (m, 3H), 2.07-0.61 (m, 13H); LCMS: [M+H] + The theoretical value of m / z is 270.1892, and the measured value is 270.1883.

[0048] 3. Synthesis of acridine orange-polythiophene fluorescent probe

[0049] 0.13 g (0.80 mmol) of ferric chloride was dissolved in 10 mL of chloroform, and 0.005 g (0.01 mmol) of TBA and 0.064 g (0.19 mmol) of TBT were dissolved in 5 mL of chloroform. The resulting chloroform solutions of TBA and TBT were then added dropwise to the chloroform solution of ferric chloride. The mixture was stirred at room temperature for 48 h under a nitrogen atmosphere. The solid obtained after rotary evaporation of the solvent was dissolved in 5 mL of methanol, reduced with 1.5 mL of hydrazine hydrate, filtered, and the filtrate was collected. Methanol was removed by rotary evaporation, and the resulting solid was dissolved in 8 mL of a 1:10 mixture of dimethyl sulfoxide and water. The solution was dialyzed through a dialysis bag with a molecular weight of 3500 for 3 days, and then lyophilized to obtain the acridine orange-polythiophene fluorescent probe P (TBA5-co-TBT95). The reaction equation is as follows:

[0050]

[0051] The theoretical ratio of the product P(TBA5-co-TBT95) is m:n = 1:19, and the experimental ratio is m:n = 1 / 2 : 97.92 / 9 = 1 : 21.76. (See...) Figure 1 . Example 2

[0052] 1. Synthesis of thiophene-acridine orange derivatives

[0053] This step is the same as step 1 in Example 1.

[0054] 2. Synthesis of thiophene quaternary ammonium salt derivatives

[0055] This step is the same as step 2 in Example 1.

[0056] 3. Synthesis of acridine orange-polythiophene fluorescent probe

[0057] 0.065 g (0.40 mmol) of ferric chloride was dissolved in 5 mL of chloroform, and 0.005 g (0.01 mmol) of TBA and 0.035 g (0.1 mmol) of TBT were dissolved in 3 mL of chloroform. The resulting chloroform solutions of TBA and TBT were then added dropwise to the chloroform solution of ferric chloride. The mixture was stirred at room temperature for 48 h under a nitrogen atmosphere. The solid obtained after rotary evaporation of the solvent was dissolved in 5 mL of methanol, reduced with 1.5 mL of hydrazine hydrate, filtered, and the filtrate was collected. Methanol was removed by rotary evaporation, and the resulting solid was dissolved in 8 mL of a 1:10 mixture of dimethyl sulfoxide and water. The solid was dialyzed through a dialysis bag with a molecular weight of 3500 for 3 days and then lyophilized to obtain the acridine orange-polythiophene fluorescent probe P (TBA10-co-TBT90).

[0058]

[0059] The theoretical ratio of the product P(TBA10-co-TBT90) is m:n = 1:9, and the experimental ratio is m:n = 1 / 2 : 45.47 / 9 = 1 : 10.10. (See...) Figure 2 .

[0060] Comparative Example 1

[0061] Following the method of Example 1, without adding acridine orange, and with all other steps the same as in Example 1, the fluorescent probe PTBT was obtained, and its NMR results are shown in [Figure 1]. Figure 3 .

[0062] Example 3

[0063] Anti-tumor recognition targeting ecDNA

[0064] Fluorescent probes PTBT, P(TBA5-co-TBT95), and P(TBA10-co-TBT90) were dissolved in water to prepare stock solutions of 20 mmol / L. The cell lines used were the colorectal cancer cell line COLO320DM containing ecDNA, the prostate cancer cell line PC3, and the colon cancer cell line HCT116 without ecDNA.

[0065] (1) The three cell lines were cultured until the confluence reached 80%–90%, digested with 0.25% trypsin, and centrifuged to prepare a cell suspension. The cells were seeded in glass-bottomed culture dishes, and after 24 h of cell growth, PTBT, P(TBA5-co-TBT95) and P(TBA10-co-TBT90) (final concentration 50 mmol / L) were added and incubated at 37 ℃ for 4 h. The cells were washed with PBS, and then PI staining solution (final concentration 50 mg / mL) was added. After washing with PBS, phenol red-free culture medium was added, and the cells were observed and photographed under a confocal microscope.

[0066] from Figures 4-6 As can be seen from the above, the fluorescent probes P(TBA5-co-TBT95) and P(TBA10-co-TBT90) of this invention can penetrate the cell membrane and enter the cell nucleus at the single-cell imaging level, bind to nucleic acids, and stain. Their fluorescence intensity is relatively strong. Furthermore, PI staining has verified that P(TBA5-co-TBT95) and P(TBA10-co-TBT90) have low cytotoxicity. PTBT, as a contrast agent, has extremely weak fluorescence intensity and cannot achieve the staining purpose.

[0067] (2) The three cell lines were cultured until the confluence reached 80%–90%, and then PTBT, P(TBA5-co-TBT95), and P(TBA10-co-TBT90) (final concentration 50 mmol / L) were added and incubated at 37°C for 4 h. The cells were digested with 0.25% trypsin, and the cell pellet was collected by centrifugation. The pellet was resuspended in 10 mM HEPES buffer (containing 0.1% serum) and incubated at 37°C for 30 min. After centrifugation, the cells were resuspended in PBS, and the cell density was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL, and each group of cell samples were analyzed using a flow cytometer.

[0068] from Figures 7-9 As can be seen from the above, the fluorescent probes P(TBA5-co-TBT95) and P(TBA10-co-TBT90) of this invention can cross the cell membrane and enter the cell nucleus at the multicellular statistical level, bind to nucleic acids, and stain, exhibiting strong fluorescence intensity. By adjusting the polymerization ratio of TBA to TBT, the cytotoxicity of the fluorescent probes can be reduced. PTBT, as a contrast agent, has extremely weak fluorescence intensity and cannot achieve the staining purpose.

[0069] (3) The three cell lines were cultured until the confluence reached 80%–90%. The cells were digested with 0.25% trypsin and centrifuged to prepare a cell suspension. The cells were seeded in 96-well plates (cell density 8000 cells / well) and incubated at 37 °C for 24 h. Three fluorescent probes, PTBT, P(TBA5-co-TBT95), and P(TBA10-co-TBT90), were added at gradient concentrations (0, 10, 20, 50, 100 mmol / L), with each concentration repeated three times in parallel, and cultured for another 24 h. 20 mL of MTT (final concentration 5 mg / mL) was added, and the plates were incubated at 37 °C for 4 h. The liquid in the wells was carefully aspirated and 150 mL of dimethyl sulfoxide was added. The 96-well plates were placed on a shaker and shaken for 10 min to ensure that the purple crystals were completely dissolved. The OD was measured using a microplate reader. 490nm The cytotoxicity of the three fluorescent probes was calculated.

[0070] Depend on Figures 10-12 As can be seen, P(TBA5-co-TBT95) was toxic to COLO320DM and PC3 after 24 h of incubation, but had no effect on HCT116, and the results for P(TBA10-co-TBT90) were the same. The difference is that P(TBA5-co-TBT95) was less toxic than P(TBA10-co-TBT90), making it more suitable for biological cell research. As a control, PTBT, which does not contain AO, had no significant effect on cell proliferation. Therefore, the fluorescent probe of this invention can inhibit the growth of cells containing ecDNA, and the growth inhibition can be regulated by different polymerization ratios of TBA and TBT.

[0071] (4) The procedure for measuring Transwell cell migration ability is as follows: Place the Transwell chamber (8 mm pore size) into a 24-well plate, add 500 mL of culture medium containing 20% ​​FBS to the lower chamber, and add 200 mL of culture medium with a density of 5×10⁻⁶ cells to the upper chamber. 5 Cell suspensions of 10 mL / mL were added to the upper and lower chambers of the experimental group, while equal volumes of deionized water were added to the upper and lower chambers of the control group. Three replicates were prepared for each group. After incubation at 37 °C for 24 h, the Transwell chambers were removed, rinsed with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 min. Following PBS rinsing, the cells were stained with 0.1% crystal violet at room temperature for 20 min, rinsed with PBS, and the cells on the bottom membrane surface of the upper chamber were carefully wiped away with cotton swabs. Cells that migrated to the lower layer of the microporous membrane were counted under an inverted microscope.

[0072] The procedure for measuring Transwell cell invasion ability is similar to that for measuring Transwell cell migration ability. The Transwell chambers need to be pre-coated with Matrigel: Take an appropriate amount of serum-free culture medium, add Matrigel at a ratio of 1:8, mix well (operate on ice), and then add it to each well of the upper chamber (50-60 μL / well). Incubate in an incubator for 4-5 h before using it for invasion experiments.

[0073] The cell scratch assay procedure involved adjusting the cell density to 5 × 10⁻⁶. 5 The cells were seeded into 6-well plates and, after complete adhesion, a horizontal line was drawn on the surface of the cell layer using a sterile pipette tip. The cells were washed with PBS, and low-serum culture medium (1% serum) was added. The experimental group contained 10 mL of P(TBA5-co-TBT95) (final concentration 50 mM), and the control group contained 10 mL of deionized water. After culturing for 24 h, the cells were observed and photographed under an inverted microscope.

[0074] Depend on Figures 13-15 As can be seen, at the 24-hour time point, the experimental group of PC3 cells filled the damaged area faster than the control group. Transwell experiments consistently showed that the number of PC3 cells migrating through the 8 μm filter was higher than that of HCT116 cells after 24 hours. Therefore, the fluorescent probe of this invention has a significant inhibitory effect on the migration and invasion ability of cells containing ecDNA.

Claims

1. An acridine orange-polythiophene fluorescent probe, characterized in that, The structural formula of the fluorescent probe is as follows: In the formula, m is an integer from 5 to 30, and n = 100 - m.

2. A method for synthesizing the acridine orange-polythiophene fluorescent probe according to claim 1, characterized in that, The method includes the following steps: (1) Synthesis of thiophene-acridine orange derivatives Acridine orange base was dissolved in toluene, heated to 70–80 °C, stirred for 30–35 min, then 3-(3-bromo)butoxy-4-methylthiophene was added, heated to 105–110 °C, refluxed under argon atmosphere for 22–24 h, washed with toluene, separated and purified to obtain the thiophene-acidine orange derivative with the following structural formula; (2) Synthesis of thiophene quaternary ammonium salt derivatives 3-(3-bromo)butoxy-4-methylthiophene and triethylamine were added to tetrahydrofuran, heated to 70-75 °C, refluxed for 46-48 h, and separated and purified to obtain a thiophene quaternary ammonium salt derivative with the following structural formula; (3) Synthesis of acridine orange-polythiophene fluorescent probe Thiophene-acridine orange derivative and thiophene quaternary ammonium salt derivative were dissolved in chloroform. The resulting solution was then added dropwise to a chloroform solution of ferric chloride. The mixture was stirred at room temperature for 46–48 h under a nitrogen atmosphere. The solvent was evaporated to dryness, and the resulting solid was dissolved in methanol. Then, hydrazine hydrate was added for reduction, and the mixture was filtered. The filtrate was collected, and methanol was removed by rotary evaporation. The resulting solid was dissolved in dimethyl sulfoxide and water, and purified by dialysis to obtain the acridine orange-polythiophene fluorescent probe.

3. The method for synthesizing the acridine orange-polythiophene fluorescent probe according to claim 2, characterized in that, In step (1), the molar ratio of acridine orange to 3-(3-bromo)butoxy-4-methylthiophene is 1:0.5 to 0.

8.

4. The method for synthesizing the acridine orange-polythiophene fluorescent probe according to claim 2, characterized in that, In step (2), the molar ratio of triethylamine to 3-(3-bromo)butoxy-4-methylthiophene is 1:26 to 28.

5. The method for synthesizing the acridine orange-polythiophene fluorescent probe according to claim 2, characterized in that: In step (3), the molar ratio of the thiophene-acridine orange derivative to the thiophene quaternary ammonium salt derivative is m:n, where m is an integer from 5 to 30 and n = 100-m.

6. The use of the acridine orange-polythiophene fluorescent probe according to claim 1 in the preparation of antitumor recognition probes targeting ecDNA.

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