CN114105967A

CN114105967A - Targeting mitochondrial AIE fluorescent probe capable of inducing tumor cell apoptosis and preparation method and application thereof

Info

Publication number: CN114105967A
Application number: CN202111282800.3A
Authority: CN
Inventors: 和庆钢; 余开武; 毛峥伟; 张浩可; 赵建江; 张宏; 田梅
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-03-01
Anticipated expiration: 2041-11-01
Also published as: CN114105967B

Abstract

The invention discloses a mitochondrial targeted aggregation-induced emission (AIE) fluorescent probe and a preparation method and application thereof, wherein the compound is marked as TPA-2TIN and has the structure shown in the specification. The AIE fluorescent probe can generate visible light fluorescence and near-infrared two-region fluorescence under the irradiation of different exciting lights, can be combined with tumor cell mitochondria through electrostatic interaction to realize mitochondrial imaging, and induces caspase-3 to activate and cut gasdermin E (GSDME) by triggering mitochondrial ROS oxidative stress, thereby further damaging cell membranes and causing tumor cell apoptosis, and thus the AIE fluorescent probe can be used as a tumor imaging probe and a therapeutic drug. In addition, the AIE fluorescent probe has photodynamic effect and photothermal effect, and can be used as a phototherapy reagent for treating tumors.

Description

Targeting mitochondrial AIE fluorescent probe capable of inducing tumor cell apoptosis and preparation method and application thereof

Technical Field

The invention belongs to the field of tumor imaging and anti-tumor treatment, and particularly relates to a targeted mitochondrial AIE fluorescent probe capable of inducing tumor cell apoptosis, and a preparation method and application thereof.

Background

Cancer is the second leading cause of death next to cardiovascular diseases in the world, and despite the great development of cancer diagnosis technology and treatment methods, tumor patients continue to grow, and the prognosis of treatment is difficult to achieve the expected effect. Therefore, the development of new tumor imaging agents and therapeutic drugs is of great significance for the early diagnosis and treatment of tumors. In recent years, the discovery and mechanism analysis of cell apoptosis provide a new scheme for anti-tumor treatment. The cell apoptosis is a programmed cell death mode, and is different from the traditional apoptosis and necrosis, the activation mode is that caspase-1/3/4/5/11 is activated to further cut GSDM D/E/A3 protein, and the cut fragments are combined with cell membranes and perforated to destroy the cell membrane potential, so that the cell swelling and vacuolation are caused, and simultaneously, the cell is caused to secrete a large amount of lactate dehydrogenase and cytokines to generate an inflammatory response and an immune stimulation effect. Therefore, the tumor scorch can produce auxiliary enhancement effect on the tumor immunotherapy.

On the other hand, the state of the position of the tumor can be visually analyzed by means of a molecular imaging means, so that tumor treatment can be guided better. Fluorescence imaging is an imaging means widely used for preclinical research, plays a certain role in clinical tumor fluorescence operation navigation, combines the functions of fluorescence imaging and tumor treatment, and develops an integrated tumor diagnosis and treatment probe, which plays an important role in accurate diagnosis and treatment of tumors. The traditional fluorescent probe is easy to generate an aggregate fluorescence quenching (ACQ) effect after aggregation occurs at a higher concentration, so that the fluorescence disappears. Therefore, the concentration of the fluorescent dye is required to be controlled in an extremely low concentration range, which in turn causes the fluorescent dye to be easy to generate photobleaching, causes the fluorescence quantum efficiency to be remarkably reduced, and greatly limits the imaging time. Aggregation-induced emission (AIE) molecules are a new class of fluorescent materials that have attracted much attention in recent years, which produce fluorescence enhancement under aggregation conditions, resist photobleaching, and thus allow imaging times to be greatly increased, allowing disease progression to be tracked more stably and over time.

At present, the AIE probe which directly causes the tumor cell apoptosis is not reported, the invention discloses a mitochondrion targeting AIE fluorescent probe and a preparation method and application thereof, and particularly the application of the probe in tumor imaging and apoptosis treatment and the combination of the probe and anti-tumor immunotherapy have wide prospects.

Disclosure of Invention

The invention aims to provide a targeted mitochondrial AIE fluorescent probe capable of inducing tumor cell apoptosis and a preparation method and application thereof.

The small molecular AIE fluorescent probe is marked as TPA-2TIN and has the following structure:

wherein the counter anion R-is selected from single-charge or multi-charge anions, and can be Cl-, Br-, I-HCO₃ ^-、PF₆ ^-、CO₃ ^2-、SO₄ ^2-、PO₄ ^3-And the like.

The invention also discloses a synthetic method of the AIE fluorescent probe, which comprises the following steps:

1) adding a compound 4-methoxyaniline with a structure shown as a formula 1 and 1-bromo-4-iodobenzene into toluene according to a molar ratio of 1: 2.2-1: 3, and reacting at 115-125 ℃ by using cuprous iodide, 1, 10-phenanthroline and potassium tert-butoxide as catalysts to obtain a compound 2;

2) dissolving a compound 2 and pinacol diboron in a molar ratio of 1: 2.2-1: 3 in 1, 4-dioxane, and reacting at the heating temperature of 105-120 ℃ by using 1,1' -bis (diphenylphosphine) ferrocene palladium dichloride and potassium acetate as catalysts to obtain a compound 3;

3) adding a compound 3 and 5-bromothiophene-2-formaldehyde into a toluene/ethanol/water mixed solution according to a molar ratio of 1: 2.2-1: 3, taking palladium tetratriphenylphosphine and potassium carbonate as catalysts, heating to 105-115 ℃, and reacting to obtain a compound 4;

4) dissolving a

compound

4, 1, 2-trimethyl-1H-benzo [ e ] indole and potassium tert-butoxide in ethanol according to a molar ratio of 1:2.5: 10-1: 10:50, heating to 75-90 ℃, and reacting to obtain a compound 5:

5) dissolving a compound 5 and methyl iodide in acetonitrile according to a molar ratio of 1: 5-1: 25, heating to 85-100 ℃, reacting to obtain an intermediate, removing the solvent, and adding NH₄R (R is selected from single-charge or multi-charge anions) and methanol/water solution, and reacting to obtain a compound TPA-2 TIN:

the AIE fluorescent probe is applied to the preparation of tumor cell imaging reagents.

The AIE fluorescent probe is applied to the preparation of tumor imaging reagents.

The AIE fluorescent probe is applied to the preparation of the medicine for triggering the tumor cell apoptosis, and the mechanism of the medicine for triggering the tumor cell apoptosis is as follows: AIE causes the oxidative stress of tumor cell mitochondria ROS, triggers caspase-3 activation and GSDME (gasdermin E) cutting, and leads to the rupture of cell membranes and finally the scorching of tumor cells.

The AIE fluorescent probe is applied to the preparation of tumor photodynamic or photothermal treatment reagents.

The invention has the beneficial effects that:

the invention provides an AIE fluorescent probe capable of directly causing the scorching of tumor cells for the first time, the AIE fluorescent probe is combined with tumor cell mitochondria through electrostatic interaction, the gathering of a large number of probes in the mitochondria causes the oxidative stress of mitochondrial ROS, the mitochondrial function is damaged, the activation of caspase-3 in cells is triggered, GSDME is cut, the cut segment is combined with the cell membranes and perforated, the cell membrane dysfunction and the cell swelling and rupture are caused, contents including lactate dehydrogenase, cell factors and the like flow out, and finally the cells die. In addition, the AIE fluorescent probe can generate blue fluorescence under the excitation of short wave, thereby realizing cell imaging and mitochondrial imaging; and near-infrared two-region fluorescence can be generated under the excitation of the long wave, so that living body fluorescence imaging can be realized. Therefore, the developed AIE fluorescent probe can be simultaneously used as an imaging reagent and a tumor treatment drug for tumor imaging and chemotherapy and immunotherapy research. In addition, the AIE probe has both photodynamic and photothermal effects, and has application value in tumor photodynamic or photothermal treatment.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings.

FIG. 1 is a synthetic roadmap for the AIE fluorescent probe TPA-2 TIN;

FIG. 2 is a drawing of Compound 2¹H NMR analysis chart;

FIG. 3 is a drawing of Compound 3¹H NMR analysis chart;

FIG. 4 is a drawing of Compound 4¹H NMR analysis chart;

FIG. 5 is a drawing of Compound 5¹H NMR analysis chart;

FIG. 6 is a diagram of the compound TPA-2TIN¹H NMR analysis chart;

FIG. 7 is an absorption spectrum of the AIE fluorescent probe;

FIG. 8 is a graph of the emission spectrum of an AIE fluorescent probe under excitation at 405 nm;

FIG. 9 is a graph of the emission spectra of AIE fluorescent probes in DMSO/Toluene solvents at different ratios under excitation at 600 nm;

FIG. 10 is a tumor cytotoxicity plot of AIE fluorescent probes;

FIG. 11 is an image taken by a microscope of the tumor cell apoptosis induced by AIE fluorescent probe (the arrows indicate the apoptotic cells);

FIG. 12 is a diagram of Annexin V/PI double positive flow quantification after incubation of AIE fluorescent probe with tumor cells;

FIG. 13 is a photograph of an immunoblot analysis of AIE fluorescent probes incubated with tumor cells;

FIG. 14 is a photograph of confocal images of the AIE fluorescent probe and the mitochondrial commercial dye MTDR after incubation with tumor cells;

FIG. 15 shows 0.1W/cm after mixing of AIE fluorescent probe and ABDA²ABDA absorption intensity change diagram under laser illumination;

FIG. 16 is a graph showing the temperature changes of the AIE fluorescent probe solution under laser irradiation of different light intensities.

Detailed Description

Materials and reagents

4-methoxyaniline, 1-bromo-4-iodobenzene, cuprous iodide, pinacol diboron, 1,1' -bisdiphenylphosphinoferrocene palladium dichloride, tetratriphenylphosphine palladium, potassium tert-butoxide, etc. were purchased from Annagi reagent. Methyl iodide, 1,1, 2-trimethyl-1H-benzo [ e ] indole, 1, 10-phenanthroline, 3, 5-bromothiophene-2-carbaldehyde and the like were purchased from the alatin reagent company. Potassium hydroxide, potassium carbonate, potassium acetate and the like are available from national chemical agents Co.

Example 1

The synthetic route of the AIE fluorescent probe of the invention is shown in figure 1, and the invention is further illustrated by combining a specific example as follows:

compound 2

Under the protection of nitrogen, 6.16g (50.0mmol) of 4-methoxyaniline, 35.40g (125.0mmol) of 1-bromo-4-iodobenzene, 2.20g (0.02mmol) of 1, 10-phenanthroline, 22.40g (0.2mmol) of potassium tert-butoxide, 1.90g (10.2mmol) of cuprous iodide are added into 85mL of toluene, reflux reaction is carried out at 116 ℃ for 13h, cooling is carried out, the solvent is removed in vacuum, dichloromethane and water are added for extraction, the organic phase is collected, dried and filtered, the solvent is removed in vacuum, and the solid is subjected to column chromatography separation to obtain 5.4g of a white solid product (yield: 25.0%).

Compound 3

Under the protection of nitrogen, 5.0g (11.5mmol) of the compound 2, 7.2g (28.2mmol) of pinacol diboron, 6.30g (64.7mmol) of potassium acetate and 0.6g (0.8mmol) of 1,1' -bis-diphenylphosphino ferrocene palladium dichloride are dissolved in 120mL of 1, 4-dioxane, refluxed for 8 hours at 110 ℃, cooled, extracted by adding dichloromethane and water, collected and dried by using anhydrous sodium sulfate. The solvent was removed in vacuo, and the oily liquid was subjected to column chromatography to give 5.4g (yield: 89.4%) of an orange-yellow oily liquid product.

Compound 4

Under the protection of nitrogen, 3.0g (5.7mmol) of the compound 3, 2.6g (13.7mmol) of 5-bromothiophene-2-carbaldehyde, 12.3g (90.0mmol) of potassium carbonate and 0.35g (0.3mmol) of tetratriphenylphosphine palladium are added into 90/45/22.5mL of a toluene/water/ethanol mixed solvent, and the mixture is refluxed for 6 hours at 110 ℃. After cooling, water and ethyl acetate were added for extraction, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and the solid was subjected to column chromatography to give 1.36g (yield: 48.3%) of a red solid product.

Compound 5

Under the protection of argon, 0.2g (0.4mmol) of compound 4, 0.5g (2.4mmol) of 1,1, 2-trimethyl-1H-benzo [ e ] indole, 1.3g (12.0mmol) of potassium tert-butoxide are dissolved in 6mL of ethanol and reacted at 80 ℃ under reflux for 12 hours. After cooling, the solvent was removed in vacuo, and the solid was subjected to column chromatography to give 0.1154g (yield: 32.8%) of a red solid product,

compound TPA-2TIN

0.04g (0.04mmol) of Compound 5, 0.11g (0.8mmol) of methyl iodide were dissolved in 3mL of acetonitrile, and the reaction was refluxed at 90 ℃ for 7 hours. Cooling, addition of ether to give a black solid, filtration and washing with ether, drying, dissolving the solid in 15mL of methanol, addition of 10mL of saturated NH₄PF₆The aqueous solution was stirred at room temperature for 24 hours, and a black solid was obtained by suction filtration and dried to obtain 0.0382g (yield: 82.2%)

Example 2

Detection of tumor cell killing effect of probe

4T1 cells are inoculated into a 96-well plate and cultured for 24h, the nano form of TPA-2TIN molecules (TPA-2TIN-NPs, a preparation method of the nano form molecules) diluted by different volumes of culture media is added, 1mg of TPA-2TIN molecules and 5mg of DSPE-PEG2k are dissolved in 1mL of DMF, 9mL of water is dropwise added under the ultrasonic condition, ultrasonic treatment is carried out for 2min, DHF is removed through dialysis, the nano form TPA-2TIN molecules are obtained), the cells are incubated for 24h at 37 ℃ in a cell incubator, then the cells are carefully washed twice by PBS, cck-8 reagent is added, the cells are incubated for 2h at 37 ℃ in the cell incubator, the absorbance of each well is measured by a microplate reader, and the cell survival rate is calculated.

The killing effect of the probe on tumor cells is shown in FIG. 10. Along with the increase of the concentration of the probe, the cell survival rate is obviously reduced, and the potential of the probe as an anti-tumor drug is reflected. The AIE fluorescent probe is positively charged, and because the tumor cell mitochondria has negative membrane potential and the potential difference is much larger than the mitochondrial membrane potential difference of normal cells, the probe is more prone to gather in the tumor cell mitochondria to cause the damage of the mitochondrial function and finally cause the apoptosis of cells, thereby showing stronger tumor cell toxicity and having smaller toxicity to the normal cells.

Example 3

Tumor cell apoptosis-inducing effect detection of probe

4T1 cells were seeded into 96-well plates, cultured for 24h, added to different volumes of media diluted nano-forms of TPA-2TIN molecules (TPA-2TIN-NPs), incubated for 4h at 37 ℃ in a cell culture incubator, and the plates were removed and photographed under a microscope.

The morphological analysis of the cells after the probe was incubated with the tumor cells is shown in FIG. 11. Along with the increase of the concentration of the probe, the tumor cells with swelling and vacuolated forms under the same size of visual field are increased, namely the number of focal death cells is continuously increased, and the medicinal effect of the probe on inducing the focal death of the tumor cells is embodied.

The results of flow quantification after probe incubation with tumor cells are shown in FIG. 12. The proportion of AM/PI double positive cells (i.e.pyrophoric cells) increased and then decreased with increasing probe concentration, reaching a maximum at 30uM, probably because further increases in concentration lead to an increase in the proportion of cells that die in other ways, such as apoptosis or necrosis, and a decrease in the proportion of pyrophoric cells.

The results of immunoblot analysis after probe incubation with tumor cells are shown in FIG. 13. Along with the increase of the concentration, the content of caspase-3 enzyme is gradually reduced, the lowest value is reached at the concentration of 30uM, 40uM is increased, the content of the GSDME protein is gradually reduced, and the content is not obviously changed in the concentration range of 20-40uM, wherein the content of a GSDME cutting fragment GSDME-N is obviously increased at the concentration of 30uM, which indicates that the proportion of coke-death cells at the concentration is higher and is consistent with the flow analysis result.

Example 4

4T1 cells were seeded into a confocal dish, incubated for 24h, added to a 1uM nano form of TPA-2TIN molecules (TPA-2TIN-NPs) diluted in medium, incubated for 4h at 37 ℃ in a cell incubator, the medium was discarded, washed three times with PBS, stained with the mitochondrial commercial dye MTDR, fixed with 4% formaldehyde, and photographed by microscopic observation.

The results of fluorescence imaging after probe incubation with cells are shown in FIG. 14. The red channel is a mitochondrion commercial dye, the green channel is a probe TPA-2TIN, and the fluorescence signals of the two dyes are highly overlapped from a fusion image, so that the targeting of the probe to the mitochondrion of the tumor cell is shown.

Example 5

ROS in vitro detection of probes

The probe prepared in example 1 (TPA-2TIN) was dissolved in DMSO at a concentration according to DMSO: water 1: 99, adding a probe into the solvent according to the volume ratio, detecting ROS by using ABDA, detecting the intensities of three characteristic absorption peaks of ABDA under an ultraviolet spectrophotometer, and when red light is used for irradiation, generating ROS by the probe to rapidly reduce the ultraviolet absorption peak of ABDA so as to perform in-vitro detection of ROS generation.

The results of the in vitro measurement of ROS production are shown in FIG. 15. When ABDA was used as an indicator, the absorption peak gradually decreased with the increase of the light irradiation time, and TPA-2TIN was found to generate ROS in vitro.

Example 6

In vitro photothermal performance detection of probe

The probe prepared in example 1 was dissolved in DMSO at a certain concentration, and the solution was irradiated with 638nm laser at different powers while monitoring the temperature change of the solution in real time with a thermal imager, and recorded every 30 seconds.

The results of photothermal effect detection of the probe are shown in FIG. 16. Along with the increase of the irradiation time, the temperature of the solution gradually rises and tends to be stable, and the laser irradiation power is increased, so that the temperature difference of the solution can be continuously increased to be 0.3W/cm²The temperature difference of 35 ℃ can be achieved under the power of the solar cell, the solar cell has stronger photothermal performance, and has potential application value in tumor photothermal treatment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. A small molecular AIE fluorescent probe is characterized in that a compound is marked as TPA-2TIN and has the following structure:

wherein, R is^-Selected from singly or multiply charged anions.

2. A method for preparing the small molecular AIE fluorescent probe of claim 1, comprising the steps of:

1) adding a compound 4-methoxyaniline and 1-bromo-4-iodobenzene with a structure shown in a formula 1 into toluene, and reacting by using cuprous iodide, 1, 10-phenanthroline and potassium tert-butoxide as catalysts to obtain a compound 2;

2) dissolving a compound 2 and pinacol diboron in 1, 4-dioxane, and reacting by using 1,1' -bis (diphenylphosphino) ferrocene palladium dichloride and potassium acetate as catalysts to obtain a compound 3;

3) adding the compound 3 and 5-bromothiophene-2-formaldehyde into a toluene/ethanol/water mixed solution, and reacting by using tetratriphenylphosphine palladium and potassium carbonate as catalysts to obtain a compound 4;

4) dissolving the compound 4, 1, 2-trimethyl-1H-benzo [ e ] indole and potassium tert-butoxide in ethanol, and reacting to obtain a compound 5:

5) dissolving compound 5 and methyl iodide in acetonitrile, reacting to obtain intermediate, removing solvent, adding NH₄R and methanol/water solution to obtain TPA-2 TIN:

3. use of the AIE fluorescent probe of claim 1 in the preparation of a tumor cell imaging agent.

4. Use of the AIE fluorescent probe of claim 1 in the preparation of a tumor imaging agent.

5. Use of the AIE fluorescent probe of claim 1 in the preparation of a medicament for triggering tumor cell apoptosis.

6. Use of the AIE fluorescent probe of claim 1 in the preparation of a photodynamic or photothermal tumor therapy agent.