Preparation of fluorescent dye with aggregation-induced emission property and application of fluorescent dye in exosome labeling and tracing

Abstract

The invention relates to the technical field of fluorescence labeling, in particular to preparation of a fluorescent dye with aggregation-induced emission property and application of the fluorescent dye in exosome labeling and tracing. The fluorescent dye with the aggregation-induced emission property, provided by the invention, can be used for efficiently marking and tracing the exosome. Meanwhile, the fluorescent dye is free of chemical corrosion and toxicity.

Description

Technical Field

[0001] This invention relates to the field of fluorescent labeling technology, and in particular to the preparation of a fluorescent dye with aggregation-induced emission properties and its application in exosome labeling and tracing. Background Technology

[0002] Exosomes are extracellular vesicles secreted by cells, with a diameter of approximately 30–150 nm. In the tumor microenvironment, exosomes are thought to play a crucial role in communication between cancer cells, thereby coordinating cancer progression, invasion, angiogenesis, and immune regulation. Given their natural origin, exosomes accumulate almost no long-term in any tissue or organ, thus exhibiting minimal or no systemic toxicity. Exosomes released by certain cells naturally possess intrinsic targeting properties. Due to their long circulating half-life, small size, low immunogenicity, and ability to preferentially target specific cell types, exosomes exhibit ideal characteristics as carriers for delivering therapeutic cargoes (such as drugs, biomolecules, or thermotherapeutic nanoparticles) to specific cells. However, currently, there are no universal and efficient methods for labeling and tracing exosomes.

[0003] Although attempts have been made to apply traditional fluorescent dyes to exosome labeling and tracing, previous studies have not yielded entirely satisfactory results. Traditional fluorescent dyes, such as DiO-type fluorescent dyes, exhibit fluorescence quenching due to the π-π stacking effect in their planar, rigid fluorophores. Therefore, the treatment of this type of luminescent molecule aims to avoid molecular aggregation as much as possible, because they only emit light in low-concentration solutions. When the concentration is too high or they are in a solid state, the large-scale aggregation of molecules leads to a gradual weakening or even disappearance of luminescence. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing a fluorescent dye with aggregation-induced emission properties and its application in exosome labeling and tracing, wherein the fluorescent dye can efficiently label and trace exosomes.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a fluorescent dye with aggregation-induced emission properties, having the structure shown in Formula 1:

[0007]

[0008] This invention also provides a method for preparing the fluorescent dye with aggregation-induced emission properties described in the above technical solution, comprising the following steps:

[0009] Compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa, dimethyldiphenylphosphineoxanthracene and a first organic solvent were mixed and subjected to a first reflux to obtain compound 2;

[0010] Compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, tetrabutylammonium bromide solution, potassium carbonate, and a second organic solvent were mixed and subjected to a second reflux to obtain compound 3.

[0011] After mixing 4-bromophenylacetonitrile, pyridine-4-boronic acid, tetrakis(triphenylphosphine)palladium and a third organic solvent, a second aqueous solution of potassium carbonate was added, and the mixture was subjected to a third reflux to obtain compound 5;

[0012] After mixing compound 3, compound 5 and a fourth organic solvent, piperidine was added dropwise and the mixture was refluxed for the fourth time to obtain compound 4.

[0013] The compound 4, iodomethane, and the fifth organic solvent were mixed and subjected to a fifth reflux to obtain the fluorescent dye with aggregation-induced emission properties.

[0014]

[0015] Preferably, the molar ratio of compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa and dimethyldiphenylphosphineoxanthracene is 1.90:2.0:0.08:2.85:0.08;

[0016] The first organic solvent includes one or more of toluene, tetrahydrofuran, dichloromethane, and methanol;

[0017] The first reflux was performed in a protective atmosphere at a temperature of 85°C for 24 hours.

[0018] Preferably, the molar ratio of tetrabutylammonium bromide to potassium carbonate in the solution of compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, and tetrabutylammonium bromide is 1.63:1.48:0.01:0.1:4;

[0019] The solvent in the tetrabutylammonium bromide solution is one or more of toluene, tetrahydrofuran, dichloromethane, and methanol; the concentration of tetrabutylammonium bromide in the tetrabutylammonium bromide solution is 5 mmol / L.

[0020] The second organic solvent includes water;

[0021] The second reflux is carried out in a protective atmosphere, and the temperature of the second reflux is 75-120°C, and the time is 8-28 hours.

[0022] Preferably, the molar ratio of 4-bromophenylacetonitrile, pyridine-4-boronic acid, and tetrakis(triphenylphosphine)palladium is 1:(1-1.1):0.01;

[0023] The third organic solvent includes one or more of tetrahydrofuran, dichloromethane, methanol, and acetonitrile;

[0024] The ratio of 4-bromophenylacetonitrile to the second potassium carbonate aqueous solution is 1 mmol: (4-5) mL, and the concentration of the second potassium carbonate aqueous solution is 1-10 mol / L.

[0025] The third reflux is performed in a protective atmosphere, with a temperature of 60–90°C and a duration of 6–12 hours.

[0026] Preferably, the molar ratio of compound 3 to compound 5 is (0.88–1):1;

[0027] Add 2 drops of piperidine for every 3 drops of 0.88 mol of the compound;

[0028] The fourth reflux is performed in a protective atmosphere, with a temperature of 70–100°C and a duration of 6–12 hours.

[0029] Preferably, the molar ratio of compound 4 to iodomethane is 1:(5-10);

[0030] The fifth organic solvent includes one or more of acetonitrile, ethanol and methanol;

[0031] The fifth reflux is performed in a protective atmosphere, with a temperature of 60–110°C and a duration of 12–24 hours.

[0032] The present invention also provides the application of the fluorescent dye with aggregation-induced emission properties described in the above technical solution or the fluorescent dye with aggregation-induced emission properties prepared by the preparation method described in the above technical solution in the preparation of exosome-labeled tracer markers.

[0033] Preferably, the concentration of the fluorescent dye with aggregation-induced emission properties in the exosome-labeled tracer is 10–15 μmol / L.

[0034] Preferably, the concentration of the fluorescent dye with aggregation-induced emission properties in the exosome-labeled tracer is 15 μmol / L.

[0035] This invention provides a fluorescent dye with aggregation-induced emission properties, having the structure shown in Formula 1:

[0036]

[0037] The fluorescent dye with aggregation-induced emission properties described in this invention exhibits high quantum yield and is positively charged overall, thus enabling efficient labeling and tracing of exosomes. Furthermore, the fluorescent dye is chemically non-corrosive and non-toxic. Attached Figure Description

[0038] Figure 1 For the TPA-Ph-SCP 1 H NMR spectrum;

[0039] Figure 2 For the TPA-Ph-SCP 13 C NMR spectrum;

[0040] Figure 3 The experimental results of application example 1 are shown in the figure;

[0041] Figure 4 The experimental results are shown in Example 2. Detailed Implementation

[0042] This invention provides a fluorescent dye with aggregation-induced emission properties, having the structure shown in Formula 1:

[0043]

[0044] The present invention also provides a method for preparing the fluorescent dye described in the above technical solution, comprising the following steps:

[0045] Compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa, dimethyldiphenylphosphineoxanthracene and a first organic solvent were mixed and subjected to a first reflux to obtain compound 2;

[0046] Compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, tetrabutylammonium bromide solution, potassium carbonate, and a second organic solvent were mixed and subjected to a second reflux to obtain compound 3.

[0047] After mixing 4-bromophenylacetonitrile, pyridine-4-boronic acid, tetrakis(triphenylphosphine)palladium and a third organic solvent, a second aqueous solution of potassium carbonate was added, and the mixture was subjected to a third reflux to obtain compound 5;

[0048] After mixing compound 3, compound 5 and a fourth organic solvent, piperidine was added dropwise and the mixture was refluxed for the fourth time to obtain compound 4.

[0049] The compound 4, iodomethane, and the fifth organic solvent were mixed and subjected to a fifth reflux to obtain the fluorescent dye;

[0050]

[0051] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0052] In this invention, the preparation process of the fluorescent dye is shown in Formula 2:

[0053]

[0054] Equation 2.

[0055] In this invention, compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa, dimethyldiphenylphosphineoxanthracene and a first organic solvent are mixed and subjected to a first reflux to obtain compound 2.

[0056] In this invention, the first organic solvent preferably includes one or more of toluene, tetrahydrofuran, dichloromethane, and methanol, more preferably toluene; when the first organic solvent is two or more of the above-mentioned specific selections, this invention does not impose any special limitation on the ratio of the above-mentioned specific substances, and they can be mixed in any ratio. In the embodiments of this invention, the first organic solvent is specifically toluene.

[0057] In this invention, the molar ratio of compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa and dimethyldiphenylphosphineoxanthracene is 1.90:2.0:0.08:2.85:0.08.

[0058] In this invention, the preferred ratio of compound 1 to the first organic solvent is 1.90 mmol:10 mL.

[0059] The present invention does not impose any special limitations on the mixing process; any process known to those skilled in the art can be used.

[0060] In this invention, the first reflux is preferably performed in a protective atmosphere, which is preferably a helium atmosphere, a nitrogen atmosphere, or an argon atmosphere, more preferably an argon atmosphere. In this invention, the temperature of the first reflux is preferably 75°C to 120°C, more preferably 85°C; the time of the first reflux is preferably 8 to 28 hours, more preferably 24 hours.

[0061] After the first reflux is completed, the present invention preferably includes sequential filtration, evaporation, and purification. The present invention does not impose any special limitations on the filtration process; any process well-known to those skilled in the art can be used. In the present invention, the evaporation method is preferably vacuum evaporation; the present invention does not impose any special limitations on the vacuum evaporation process; any process well-known to those skilled in the art can be used. In the present invention, the purification method is preferably silica gel column chromatography; the mobile phase used in silica gel column chromatography is preferably petroleum ether, and the eluent is preferably diethyl ether / dichloromethane (the volume ratio of diethyl ether to dichloromethane is preferably 4:1).

[0062] After obtaining compound 2, the present invention mixes compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, tetrabutylammonium bromide solution, potassium carbonate, and a second organic solvent, and performs a second reflux to obtain compound 3.

[0063] In this invention, the molar ratio of tetrabutylammonium bromide and potassium carbonate in the solution of compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, and tetrabutylammonium bromide (TBAB) is preferably 1.63:1.48:0.01:0.1:4.

[0064] In this invention, the solvent in the tetrabutylammonium bromide solution is preferably one or more of toluene, tetrahydrofuran, dichloromethane and methanol, more preferably; the concentration of tetrabutylammonium bromide in the tetrabutylammonium bromide solution is preferably 5 mmol / L.

[0065] In this invention, the second organic solvent preferably includes water, and the volume ratio of the tetrabutylammonium bromide solution to the second organic solvent is preferably 10:1.

[0066] In this invention, the second reflux is preferably carried out in a protective atmosphere, which preferably includes an argon atmosphere, a nitrogen atmosphere, or a helium atmosphere, more preferably an argon atmosphere; the temperature of the second reflux is preferably 75°C to 120°C, more preferably 75°C; the time of the second reflux is preferably 8 to 28 hours, more preferably 24 hours.

[0067] After the second reflux is completed, the present invention preferably further includes separating the organic phase and the aqueous phase to obtain an aqueous phase and an organic phase; after obtaining the aqueous phase, the present invention preferably includes sequentially extracting and drying the aqueous phase; the extractant used for extraction is preferably dichloromethane, and the number of extractions is preferably 3; the drying is preferably performed using anhydrous sodium sulfate; after obtaining the organic phase, the present invention preferably performs vacuum evaporation and silica gel column chromatography purification sequentially; the eluent used for silica gel column chromatography purification is preferably petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0068] The preparation method of the present invention further includes mixing 4-bromophenylacetonitrile, pyridine-4-boronic acid, tetrakis(triphenylphosphine)palladium and a third organic solvent, adding a second potassium carbonate aqueous solution, and performing a third reflux to obtain compound 5.

[0069] In this invention, the third organic solvent preferably includes one or more of tetrahydrofuran (THF), dichloromethane, methanol, and acetonitrile; more preferably, it includes tetrahydrofuran; when the third organic solvent is two or more of the above-mentioned specific selections, this invention does not impose any special limitation on the ratio of the above-mentioned specific substances, and they can be mixed in any ratio.

[0070] In this invention, the molar ratio of 4-bromophenylacetonitrile, pyridine-4-boronic acid and tetrakis(triphenylphosphine)palladium is preferably 1:1 to 1.1:0.01, more preferably 1:1.1:0.01.

[0071] In this invention, the preferred ratio of 4-bromophenylacetonitrile to the third organic solvent is 1 mmol:(6-10) mL, more preferably 2 mmol:12 mL.

[0072] In this invention, the preferred ratio of the amount of 4-bromophenylacetonitrile to the amount of the second potassium carbonate aqueous solution is 1 mmol:(4-5) mL, more preferably 1 mmol:4 mL; the preferred concentration of the second potassium carbonate aqueous solution is 1-10 mol / L, more preferably 2 mol / L.

[0073] The present invention does not impose any special limitations on the process of mixing and adding the second potassium carbonate aqueous solution; any process known to those skilled in the art can be used.

[0074] In this invention, the third reflux is preferably carried out in a protective atmosphere, which is preferably a nitrogen atmosphere, an argon atmosphere, or a helium atmosphere, more preferably a nitrogen atmosphere; the temperature of the third reflux is preferably 60-90°C, more preferably 75°C; and the time of the third reflux is preferably 6-12 hours, more preferably 8 hours.

[0075] After the third reflux is completed, the present invention preferably includes sequential cooling, extraction of the organic phase, rotary evaporation, and silica gel column chromatography. The present invention does not impose any special limitations on the cooling process; any process well-known to those skilled in the art can be used. In the present invention, the extractant used for extracting the organic phase is preferably a saturated sodium chloride aqueous solution; the number of extractions of the organic phase is preferably three, and the amount of saturated sodium chloride aqueous solution used for each extraction is preferably 5 mL. The present invention does not impose any special limitations on the rotary evaporation and silica gel column chromatography processes; any process well-known to those skilled in the art can be used.

[0076] After obtaining compounds 3 and 5, the present invention mixes compounds 3 and 5 with a fourth organic solvent, adds piperidine dropwise, and performs a fourth reflux to obtain compound 4.

[0077] In this invention, the molar ratio of compound 3 to compound 5 is preferably (0.88:1):1, more preferably 0.88:1. For every 0.88 mol of compound 3, preferably 2 drops of piperidine are added.

[0078] In this invention, the fourth organic solvent preferably includes one or more of anhydrous ethanol, anhydrous methanol, and anhydrous acetonitrile; more preferably, it includes anhydrous ethanol; when the fourth organic solvent is two or more of the above-mentioned specific selections, this invention does not impose any special limitation on the ratio of the above-mentioned specific substances, and they can be mixed in any ratio.

[0079] The present invention does not impose any special limitations on the mixing process, and any process known to those skilled in the art can be used; the present invention also does not impose any special limitations on the method of adding piperidine, and any method known to those skilled in the art can be used.

[0080] In this invention, the fourth reflux is preferably performed in a protective atmosphere, which is preferably a nitrogen atmosphere, an argon atmosphere, or a helium atmosphere; the temperature of the fourth reflux is preferably 70-100°C, more preferably 80°C; and the time of the fourth reflux is preferably 6-12 hours, more preferably 8 hours.

[0081] After the fourth reflux is completed, the present invention preferably includes sequential cooling, rotary evaporation and silica gel column chromatography; the present invention does not impose any special limitations on the cooling, rotary evaporation and silica gel column chromatography process, and any process known to those skilled in the art can be used.

[0082] After obtaining compound 4, the present invention mixes compound 4, iodomethane and a fifth organic solvent, and performs a fifth reflux to obtain the fluorescent dye.

[0083] In this invention, the molar ratio of compound 4 to iodomethane is preferably 1:(5-10), more preferably 1:10.

[0084] In this invention, the fifth organic solvent preferably includes one or more of acetonitrile, ethanol and methanol, more preferably acetonitrile; when the fifth organic solvent is two or more of the above-mentioned specific selections, this invention does not impose any special limitation on the ratio of the above-mentioned specific substances, and they can be mixed in any ratio.

[0085] The present invention does not impose any special limitation on the amount of the fifth organic solvent; any amount known to those skilled in the art that can ensure the smooth progress of the reaction can be used.

[0086] In this invention, the fifth reflux is preferably performed in a protective atmosphere, which is preferably a nitrogen atmosphere, an argon atmosphere, or a helium atmosphere, more preferably a nitrogen atmosphere; the temperature of the fifth reflux is preferably 60-110°C, more preferably 80°C; and the time of the fifth reflux is preferably 12-24 hours, more preferably 16 hours.

[0087] After the fifth reflux is completed, the present invention preferably includes sequentially performing a first rotary evaporation, silica gel column chromatography separation, extraction, and a second rotary evaporation. The present invention does not impose any special limitations on the processes of the first rotary evaporation, silica gel column chromatography separation, and the second rotary evaporation; any process well-known to those skilled in the art can be used. In the present invention, the extraction process preferably involves dissolving the powder obtained after silica gel column chromatography in methanol, adding an equal volume of potassium hexafluorophosphate aqueous solution (concentration 2M), magnetically stirring for 4 hours, adding 20 mL of dichloromethane for extraction, and repeating the above process of adding 20 mL of dichloromethane twice, retaining the organic phase.

[0088] The present invention also provides the application of the fluorescent dye with aggregation-induced emission properties described in the above technical solution or the fluorescent dye with aggregation-induced emission properties prepared by the preparation method described in the above technical solution in the preparation of exosome-labeled tracer markers.

[0089] In this invention, the concentration of the fluorescent dye with aggregation-induced emission properties in the exosome-labeled tracer is preferably 10-15 μmol / L, more preferably 15 μmol / L.

[0090] In this invention, the solvent for the exosome-labeled tracer is preferably PBS buffer; the pH of the PBS buffer is preferably 7.2 to 7.4.

[0091] In this invention, the method for exosome labeling and tracing preferably includes the following steps:

[0092] The fluorescent dye with aggregation-induced emission (AIE), exosomes (EVs), and PBS buffer were mixed, incubated for the first time, centrifuged, and the AIE-EVs at the bottom of the tube were collected.

[0093] The AIE-EVs were resuspended in PBS buffer to obtain the culture medium;

[0094] 4T1 cells were cultured in the culture medium and then subjected to a second incubation.

[0095] In this invention, the fluorescent dye with aggregation-induced emission properties, exosomes, and PBS buffer are mixed, incubated for the first time, centrifuged, and the AIE-EVs at the bottom of the tube are collected.

[0096] In this invention, the preferred ratio of exosomes to PBS buffer is 200 μg: 1 mL. The pH of the PBS buffer is preferably 7.2–7.4, more preferably 7.2.

[0097] In this invention, the concentration of the fluorescent dye with aggregation-induced emission properties in the mixture obtained after mixing is preferably 4 μM.

[0098] The present invention does not impose any special limitations on the mixing process; any process known to those skilled in the art can be used.

[0099] In this invention, the temperature of the first incubation is preferably 34°C to 40°C, more preferably 37°C; the incubation time is preferably 1h to 8h, more preferably 2h.

[0100] In this invention, the centrifugation speed is preferably 8000g to 120000g, more preferably 10000g, and the centrifugation time is preferably 1h to 3h, more preferably 1.5h. In this invention, the centrifugation is used to remove unbound fluorescent dyes.

[0101] After obtaining the AIE-EVs, the present invention resuspends the AIE-EVs in PBS buffer to obtain the culture medium.

[0102] In this invention, the pH value of the PBS buffer is preferably 7.2 to 7.4, more preferably 7.2.

[0103] In this invention, the concentration of AIE-EVs in the culture medium is preferably 1 to 16 μM, more preferably 16 μM.

[0104] After obtaining the culture medium, the present invention uses the culture medium to culture 4T1 cells for a second incubation.

[0105] In this invention, the AIE-EVs in the culture medium for culturing 4T1 cells are preferably 80 μg. This invention does not impose any special limitations on the process of culturing 4T1 cells or the process of the second incubation; any process well-known to those skilled in the art can be used.

[0106] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0107] Example 1

[0108] The preparation process was as shown in Formula 2: Compound 1 (473 mg, 1.90 mmol), 1-bromo-4-iodobenzene (566 mg, 2.00 mmol), Pd(OAc)2 (18 mg, 0.08 mmol), t-BuONa (273 mg, 2.85 mmol) and dimethyldiphenylphosphineoxanthracene (46 mg, 0.08 mmol) were dissolved in 10 mL of toluene and refluxed at 85 °C for 24 h under an argon atmosphere. The reaction mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether using diethyl ether / dichloromethane (4:1, v / v) as the eluent to obtain compound 2.

[0109] A mixture (20 mL) of compound 2 (657 mg, 1.63 mmol), 4-formylphenylboronic acid (223 mg, 1.48 mmol), Pd(PPh3)4 (11.5 mg, 0.01 mmol), K2CO3 (552 mg, 4 mmol), and TBAB (32 mg, 0.1 mmol) and toluene, along with water (2 mL), was heated at reflux temperature (75 °C) for 24 h under an argon atmosphere. The reaction mixture was then separated. The aqueous phase was extracted three times with dichloromethane, and the organic layer was dried over Na2SO4 to give compound 3. The organic phase was evaporated under vacuum, and the crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (10:1, v / v) as eluent to give compound 3.

[0110] 4-Bromophenylacetonitrile (392 mg, 2 mmol), pyridine-4-boronic acid (271 mg, 2.2 mmol), and tetrakis(triphenylphosphine)palladium (24 mg, 0.02 mmol) were dissolved in 12 mL of THF solution. Then, 8 mL of 2 mol / L K₂CO₃ aqueous solution (containing 2.2 g of potassium carbonate) was added to the reaction flask. The mixture was refluxed at 75 °C under nitrogen protection for 8 hours. After the reaction was complete, the mixture was allowed to return to room temperature. Next, the mixture was extracted (3 times with 5 mL of saturated NaCl aqueous solution), leaving the organic phase. The organic phase was then rotary evaporated to obtain the crude product. Finally, the crude product was purified by silica gel column chromatography to obtain compound 5.

[0111] Compound 3 (416 mg, 0.88 mmol) and compound 5 (194 mg, 1 mmol) were dissolved in 5 mL of anhydrous ethanol. Then, 2 drops of piperidine were added to the mixed solution, and the mixture was refluxed at 80 °C for 8 h under nitrogen protection. After the reaction was completed, the entire reaction system was brought back to room temperature. Finally, the crude product was obtained by rotary evaporation and purified by silica gel column chromatography to obtain compound 4.

[0112] Compound 4 (415 mg, 0.64 mmol) and iodomethane (200 μL) were refluxed in acetonitrile at 80 °C for 16 h under nitrogen protection. After the reaction was complete, the crude product was obtained by rotary evaporation. The crude product was then purified by silica gel column chromatography to obtain a powder, which was dissolved in 10 mL of methanol (MeOH) solution. An equal volume of potassium hexafluorophosphate (KPF6) aqueous solution was added, and the mixture was stirred on a magnetic stirrer at room temperature for 4 h. Finally, dichloromethane (20 mL × 3) was added for extraction, and the organic phase was retained and rotary evaporated to obtain a fluorescent dye with aggregation-induced emission properties (denoted as TPA-Ph-SCP).

[0113] Figure 1 For the TPA-Ph-SCP 1 H NMR spectrum Figure 2 For the TPA-Ph-SCP 13 C NMR spectrum, by Figures 1-2 It is known that fluorescent dyes with aggregation-induced emission properties have the structure shown in Formula 1.

[0114] Application Example 1

[0115] PBS group: 4T1 cells were cultured directly in PBS buffer (pH 7.2) and incubated, followed by fluorescence imaging. The results are shown below. Figure 3 As shown;

[0116] Experimental group 1: 1.5 μL of 4 μM TPA-Ph-SCP PBS buffer (pH 7.2) and 200 μg of exosomes were dissolved in 1 mL of PBS buffer (pH 7.2) and incubated at 37 °C for 2 h to obtain a mixture;

[0117] The mixture was centrifuged for 1.5 h to remove unbound fluorescent dye (TPA-Ph-SCP). The AIE-EVs accumulated at the bottom of the tube were resuspended in PBS buffer (pH 7.2) to obtain a resuspension (concentration 3 μM).

[0118] 4T1 cells were recultured in the culture medium containing the resuspension and incubated for a further period. Fluorescence imaging was then performed, and the results are as follows: Figure 3 As shown;

[0119] Experimental group 2: 1 μL of 4 μM TPA-Ph-SCP PBS buffer (pH 7.2) and 200 μg of exosomes were dissolved in 1 mL of PBS buffer (pH 7.2) and incubated at 37 °C for 2 h to obtain a mixture;

[0120] The mixture was centrifuged for 1.5 h to remove unbound fluorescent dye (TPA-Ph-SCP). The AIE-EVs accumulated at the bottom of the tube were resuspended in PBS buffer (pH 7.2) to obtain a resuspension (concentration 2 μM).

[0121] 4T1 cells were recultured in the culture medium containing the resuspension and incubated for a further period. Fluorescence imaging was then performed, and the results are as follows: Figure 3 As shown;

[0122] Depend on Figure 3 It is known that AIE can label exosomes, and AIE-Evs can be taken up by tumor cells and have tracking potential.

[0123] Application Example 2

[0124] A tumor model was established in the left axilla of mice using mouse breast cancer 4T1 cells: 1 mL of PBS suspension of 4T1 cells (10,000 cells / mL) was injected into the left axilla of mice. After one week, the tumor volume in the axilla of the mice reached 100 mm. 3 Modeling was completed immediately, and tumor-bearing mice were obtained. The control group consisted of tumor tissue sections from tumor-bearing mice treated with PBS buffer solution. Experimental group 1 consisted of tumor tissue sections from tumor-bearing mice injected with AIE-EVs (80 mg / mL) solution, and experimental group 2 consisted of tumor tissue sections from tumor-bearing mice injected with AIE-EVs (40 mg / mL) solution.

[0125] AIE-EVs were resuspended in 100 μL of PBS buffer (pH 7.2) and administered intravenously to the experimental groups. Vital organs were collected after 48 h, processed, and subjected to fluorescence imaging. The results are shown below. Figure 4 As shown (AIE is green, DAPI is blue, MERGE is fluorescence overlap), by Figure 4 It can be seen that, compared with untreated mice, the tumor sites of treated mice showed obvious and strong fluorescence signals, proving that AIE-EVs have good tumor targeting effects and concentration-dependent fluorescence imaging capabilities at the tumor sites.

[0126] In summary, the fluorescent dye with aggregation-induced emission properties described in this invention is effective as an exosome labeling dye, and the labeling effect is most significant at a concentration of 15 μM; it also has significant effects in tumor labeling and tracing.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent dye with aggregation-induced emission properties, characterized in that, It has the structure shown in Equation 1:

2. The method for preparing the fluorescent dye with aggregation-induced emission properties according to claim 1, characterized in that, Includes the following steps: Compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa, dimethyldiphenylphosphineoxanthracene and a first organic solvent were mixed and subjected to a first reflux to obtain compound 2; The compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, tetrabutylammonium bromide solution, potassium carbonate, and a second organic solvent were mixed and subjected to a second reflux to obtain compound 3. After mixing 4-bromophenylacetonitrile, pyridine-4-boronic acid, tetrakis(triphenylphosphine)palladium and a third organic solvent, a second aqueous solution of potassium carbonate was added, and the mixture was subjected to a third reflux to obtain compound 5; After mixing compound 3, compound 5 and a fourth organic solvent, piperidine was added dropwise and the mixture was refluxed for the fourth time to obtain compound 4. The compound 4, iodomethane, and the fifth organic solvent were mixed and subjected to a fifth reflux to obtain the fluorescent dye with aggregation-induced emission properties.

3. The preparation method according to claim 2, characterized in that, The molar ratio of compound 1, 1-bromo-4-iodobenzene, Pd(OAc)2, t-BuONa, and dimethyldiphenylphosphineoxanthracene is 1.90:2.0:0.08:2.85:0.08; The first organic solvent includes one or more of toluene, tetrahydrofuran, dichloromethane, and methanol; The first reflux is performed in a protective atmosphere, and the temperature of the first reflux is 75-120°C, and the time is 8-28 hours.

4. The preparation method according to claim 2, characterized in that, The molar ratio of tetrabutylammonium bromide to potassium carbonate in the solution of compound 2, 4-formylphenylboronic acid, Pd(PPH3)4, and tetrabutylammonium bromide is 1.63:1.48:0.01:0.1:4; The solvent in the tetrabutylammonium bromide solution is one or more of toluene, tetrahydrofuran, dichloromethane, and methanol; the concentration of tetrabutylammonium bromide in the tetrabutylammonium bromide solution is 5 mmol / L. The second organic solvent includes water; The second reflux is carried out in a protective atmosphere, and the temperature of the second reflux is 75-120°C, and the time is 8-28 hours.

5. The preparation method according to claim 2, characterized in that, The molar ratio of 4-bromophenylacetonitrile, pyridine-4-boronic acid and tetrakis(triphenylphosphine)palladium is 1:(1-1.1):0.01; The third organic solvent includes one or more of tetrahydrofuran, dichloromethane, methanol, and acetonitrile; The ratio of 4-bromophenylacetonitrile to the second potassium carbonate aqueous solution is 1 mmol: (4-5) mL, and the concentration of the second potassium carbonate aqueous solution is 1-10 mol / L. The third reflux is performed in a protective atmosphere, with a temperature of 60–90°C and a duration of 6–12 hours.

6. The preparation method according to claim 2, characterized in that, The molar ratio of compound 3 to compound 5 is (0.88–1):1; Add 2 drops of piperidine for every 3 drops of 0.88 mol of the compound; The fourth reflux is performed in a protective atmosphere, with a temperature of 70–100°C and a duration of 6–12 hours.

7. The preparation method according to claim 2, characterized in that, The molar ratio of compound 4 to iodomethane is 1:(5-10); The fifth organic solvent includes one or more of acetonitrile, ethanol and methanol; The fifth reflux is performed in a protective atmosphere, with a temperature of 60–110°C and a duration of 12–24 hours.

8. The application of the fluorescent dye with aggregation-induced emission properties as described in claim 1 or the fluorescent dye with aggregation-induced emission properties prepared by the preparation method described in any one of claims 2 to 7 in the preparation of exosome-labeled tracers.

9. The application as described in claim 8, characterized in that, The concentration of the fluorescent dye with aggregation-induced emission properties in the exosome-labeled tracer is 10–15 μmol / L.

10. The application as described in claim 8, characterized in that, The concentration of the fluorescent dye with aggregation-induced emission properties in the exosome-labeled tracer is 15 μmol / L.

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