Fluorescent compounds, methods of making and using the same, and methods of labeling extracellular vesicles

Abstract

The application provides a fluorescent compound, a preparation method and application thereof, and a method for labeling extracellular vesicles, and belongs to the technical field of biological medicines. The fluorescent compound provided by the application comprises a cationic group and an anionic group, the anionic group is selected from iodine ions, bromine ions or PF6 ‑ , and the cationic group has a structure shown in formula I. The fluorescent compound provided by the application has high extracellular vesicle labeling efficiency, and the labeled extracellular vesicles have high fluorescence intensity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a fluorescent compound and a preparation method and application thereof, and a method for labeling extracellular vesicles. BACKGROUND

[0002] Before extracellular vesicle preparations are applied to clinical treatment of diseases, the biological behavior of the extracellular vesicles after entering the human body must be understood to determine the safety thereof. Therefore, mastering mature extracellular vesicle tracing technology is of great significance for promoting the clinical conversion of extracellular vesicle treatment of diseases. At present, the most commonly used commercial extracellular vesicle tracing probe in the academic circle is a lipophilic fluorescent probe, such as PKH26. However, the extracellular vesicle labeling efficiency of this kind of probe and the fluorescence intensity of the labeled extracellular vesicles still need to be improved. SUMMARY

[0003] The present application relates to the technical field of biological medicine, and particularly relates to a fluorescent compound and a preparation method and application thereof, and a method for labeling extracellular vesicles.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0005] The present application provides a fluorescent compound, which comprises a cationic group and an anionic group, the anionic group is selected from iodine ion, bromine ion or PF6 - , and the cationic group has the structure shown in formula I:

[0006]

[0007] Preferably, the anionic group comprises PF6 - , bromine ion or iodine ion.

[0008] The present application provides a preparation method of the fluorescent compound in the above technical solution, which comprises the following steps:

[0009] 5-[4-(diphenyl-amino)phenyl]thiophene-2-ethanal, 1,2,6-trimethylpyridine iodide, an acid binding agent and an organic solvent are mixed to perform a condensation reaction, so as to obtain a fluorescent compound with iodine ion as the anionic group;

[0010] The fluorescent compound with iodine ion as the anionic group is mixed with an anionic group donor, an organic solvent and water to perform a replacement treatment, so as to obtain the fluorescent compound with bromine ion or PF6 - as the anionic group; wherein the anionic group in the anionic group donor is bromine ion or PF6 - .

[0011] Preferably, the acid-binding agent comprises piperidine, sodium ethoxide or sodium methoxide.

[0012] Preferably, the temperature of the condensation reaction is the reflux temperature of the system, and the time of the condensation reaction is 10-15 h.

[0013] Preferably, the anion group donor comprises KPF6 or KBr; the temperature of the displacement treatment is 20-30℃, and the time of the displacement treatment is 3-5 h.

[0014] The application provides application of the fluorescent compound in the preparation of an extracellular vesicle tracing reagent.

[0015] The application provides a method for labeling extracellular vesicles, comprising the following steps:

[0016] The probe is the fluorescent compound in the above technical solution.

[0017] Preferably, the extracellular vesicles comprise human umbilical vein endothelial cell extracellular vesicles; and the use ratio of the probe to the extracellular vesicles is 4 μmol: 0.05-0.15 g.

[0018] Preferably, the dispersing agent comprises an organic solvent and a buffer solution; the mixing comprises: first mixing the probe and the organic solvent to obtain a probe mother liquor; second mixing the extracellular vesicles and part of the buffer solution to obtain an extracellular vesicle suspension; and third mixing the probe mother liquor, the extracellular vesicle suspension and the remaining buffer solution.

[0019] Preferably, the temperature of the incubation is 35-37℃, and the time of the incubation is 1.5-2.5 h; and the incubation is carried out in the dark.

[0020] Beneficial effects: The fluorescent compound (denoted as compound P2T) provided by the application has a positive charge, the membrane surface of the extracellular vesicles has a negative charge, the compound P2T is combined with the membrane surface of the extracellular vesicles through positive-negative charge adsorption, so that the compound P2T has high labeling efficiency for the extracellular vesicles. Meanwhile, the compound P2T provided by the application has the property of aggregation-induced emission (AIE), that is, the compound P2T can emit strong fluorescence in the aggregated state, so that the extracellular vesicles labeled by the compound P2T have high fluorescence intensity. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A flow chart for preparing the compound P2T;

[0022] Figure 2 A nuclear magnetic resonance hydrogen spectrum of the compound P2T;

[0023] Figure 3 The carbon NMR spectrum of compound P2T;

[0024] Figure 4 Transmission electron micrographs of unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T;

[0025] Figure 5 Figure 1 shows the ZETA potential test results for unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T.

[0026] Figure 6 Figure 1 shows the particle size distribution of unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T.

[0027] Figure 7 The figure shows the results of the extracellular vesicle labeling efficiency test of compound P2T and probe PKH26;

[0028] Figure 8 Laser confocal microscopy images of P2T-EVs and PKH26-EVs co-cultured with the HT22 neuronal cell line and stained.

[0029] Figure 9 This is a comparison chart of fluorescence signal intensity. Detailed Implementation

[0030] This invention provides a fluorescent compound comprising a cationic group and an anionic group, wherein the anionic group is selected from iodide ions, bromide ions, or PF6. - The cationic group has the structure shown in Formula I:

[0031]

[0032] In this invention, the anionic group of the fluorescent compound is selected from PF6. - bromide or iodide ions; using the above-mentioned anionic groups is beneficial to reduce the toxicity of fluorescent compounds and improve biocompatibility.

[0033] Currently, the most commonly used commercial extracellular vesicle tracking probes are lipophilic fluorescent probes, such as PKH26. However, the labeling efficiency of these probes and the fluorescence intensity of the labeled extracellular vesicles still need improvement. Furthermore, lipophilic fluorescent probes exhibit a strong quenching effect in the aggregated state, resulting in a relatively short tracking time for extracellular vesicles. In addition, these probes affect the particle size of extracellular vesicles, thereby influencing cellular uptake of extracellular vesicles and their half-life in vivo. Therefore, developing extracellular vesicle tracking probes with excellent tracking capabilities is of great significance.

[0034] Aggregation-induced emission (AIE) materials show great potential in the field of biological imaging, and AIE fluorescent compounds and nanoparticles formed by the same have the advantages of high brightness, low background noise, uniform light stability, good biocompatibility, excellent tissue penetration ability and higher spatial resolution, and development of new extracellular vesicle tracing probes with AIE properties is expected to solve the technical difficulties in the field of in vivo tracing of extracellular vesicles. Based on the problems existing in the current extracellular vesicle tracing technology, a fluorescent compound (i.e. compound P2T) is designed, which can be used as an AIE extracellular vesicle tracing probe, providing a new method for in vivo tracing of extracellular vesicles and providing strong support for further clinical translation research of extracellular vesicles. The test results show that, compared with the commonly used commercial extracellular vesicle tracing probe PKH26, the compound P2T in the present application has higher extracellular vesicle labeling efficiency, because the compound P2T has a positive charge, and the surface of the extracellular vesicle membrane has a negative charge, so the compound P2T and the surface of the extracellular vesicle membrane are combined together by positive and negative charge adsorption. At the same time, the extracellular vesicles labeled by the compound P2T in the present application have higher fluorescence intensity, because the compound P2T has the property of aggregation-induced emission (AIE), i.e. the compound P2T can emit strong fluorescence in the aggregated state, and the tracing of extracellular vesicles lasts for a long time. In addition, the compound P2T in the present application does not affect the particle size of the extracellular vesicles, and thus does not affect the uptake of the extracellular vesicles by cells and the half-life of the extracellular vesicles in the body.

[0035] The present application provides a preparation method of the fluorescent compound described in the above technical solution, comprising the following steps:

[0036] 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridine iodide, an acid binding agent and an organic solvent are mixed to carry out a condensation reaction, so as to obtain a fluorescent compound with an anion group of iodine ion;

[0037] The fluorescent compound with the anion group of iodine ion is mixed with an anion group donor, an organic solvent and water to carry out displacement treatment, so as to obtain the fluorescent compound with the anion group of bromine ion or PF6 - . - .

[0038] In the present application, unless otherwise specified, the raw materials used are commercially available or prepared by methods well known to those skilled in the art.

[0039] This invention involves mixing 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridine iodide, an acid-binding agent, and an organic solvent to undergo a condensation reaction, yielding a fluorescent compound with an iodide ion as the anionic group. As an embodiment of this invention, the molar ratio of 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde to 1,2,6-trimethylpyridine iodide can be 2:0.8 to 1.2, specifically 2:1. As an embodiment of this invention, the acid-binding agent may include piperidine, sodium ethoxide, or sodium methoxide, and the organic solvent may be an alcohol solvent, specifically ethanol. This invention does not have specific limitations on the amount of the acid-binding agent and the organic solvent, as long as the reaction proceeds smoothly.

[0040] In one embodiment of the present invention, the temperature of the condensation reaction can be the reflux temperature of the system. Taking ethanol as an example, the temperature of the condensation reaction can be 80°C; the time of the condensation reaction can be 10-15 hours, specifically 12 hours; the condensation reaction can be carried out in a protective atmosphere, specifically an argon atmosphere. In another embodiment of the present invention, the condensation reaction preferably further includes: cooling and filtering the product system obtained after the condensation reaction, and the resulting filter cake is a fluorescent compound with an iodide ion anionic group.

[0041] After obtaining a fluorescent compound with an iodide ion as the anionic group, the present invention mixes the fluorescent compound with the iodide ion as the anionic group donor, an organic solvent, and water, and performs a displacement treatment to obtain a compound with a bromide ion or PF6 anionic group. - The fluorescent compound; wherein the anionic group donor is a bromide ion or PF6. - In one embodiment of the present invention, the anionic donor may include KPF6 or KBr; the organic solvent may be acetone, and the volume ratio of the organic solvent to water may be 1:0.8 to 1.2, specifically 1:1; the amount of the anionic donor used is based on ensuring that it forms a saturated solution with water; the present invention does not have a specific limitation on the specific amount of the organic solvent and water, as long as it can ensure that the displacement treatment proceeds smoothly. In this embodiment of the present invention, specifically, the fluorescent compound with iodide ion as the anionic group is dissolved in an organic solvent, and then an equal volume of saturated anionic donor aqueous solution is added for displacement treatment.

[0042] As an embodiment of the present application, the temperature of the displacement treatment can be 20-30℃, and specifically can be room temperature (25℃). The time of the displacement treatment can be 3-5h, and specifically can be 4h. The displacement treatment can be carried out under stirring. As an embodiment of the present application, the displacement treatment preferably further comprises: vacuum evaporation of the mixture obtained after the displacement treatment to remove the organic phase, and then silica gel column chromatography purification of the obtained crude product to obtain the fluorescent compound with an anion group of bromide or PF6 - The eluent used in the silica gel column chromatography purification can be dichloromethane and methanol, and the volume ratio of the dichloromethane and methanol can be 20:1.

[0043] The present application provides an application of the fluorescent compound in the above technical solution in the preparation of an extracellular vesicle tracing reagent.

[0044] The present application provides a method for labeling extracellular vesicles, comprising the following steps:

[0045] The probe is the fluorescent compound in the above technical solution.

[0046] As an embodiment of the present application, the extracellular vesicles can include human umbilical vein endothelial cell extracellular vesicles. In the embodiment of the present application, the human umbilical vein endothelial cell extracellular vesicles are taken as an example, and specifically, human umbilical vein endothelial cells are cultured in vitro, and cell supernatant is collected; the cell supernatant is placed in a centrifuge, centrifuged at 500g for 10min, and the precipitate is removed, and then the obtained supernatant is centrifuged at 2000g for 20min, and the precipitate is removed, and finally the obtained supernatant is centrifuged at 5000g for 30min, and the precipitate is removed; the obtained supernatant is filtered through a 0.2μm filter, and then an ultracentrifuge is used to centrifuge at 130000g for 2h, the supernatant is discarded, the precipitate is resuspended with PBS, and then the ultracentrifuge is used to centrifuge at 130000g for 2h, the supernatant is discarded, and the obtained precipitate is human umbilical vein endothelial cell extracellular vesicles.

[0047] As an embodiment of the present application, the ratio of the amount of the probe to the amount of the extracellular vesicles can be 4μmol:0.05-0.15g, and specifically can be 4μmol:0.05g, 4μmol:0.08g, 4μmol:0.1g, 4μmol:0.12g or 4μmol:0.15g.

[0048] In one embodiment of the present invention, the dispersant may include an organic solvent and a buffer solution, wherein the organic solvent may specifically be dimethyl sulfoxide (DMSO) and the buffer solution may specifically be PBS. In another embodiment of the present invention, the mixing may include: first mixing the probe with the organic solvent to obtain a probe stock solution; second mixing the extracellular vesicles with a portion of the buffer solution to obtain an extracellular vesicle suspension; and third mixing the probe stock solution, the extracellular vesicle suspension, and the remaining buffer solution. In another embodiment of the present invention, the concentration of the probe stock solution may be 4–6 mmol / L, specifically 5 mmol / L; the concentration of the extracellular vesicle suspension may be 0.5–2.0 g / L, specifically 1.0 g / L; and the volume ratio of the probe stock solution, the extracellular vesicle suspension, and the remaining buffer solution may be 0.8:100:899.2.

[0049] As an embodiment of the present invention, the incubation temperature can be 35-37°C, the incubation time can be 1.5-2.5 hours, specifically 2 hours; the incubation is preferably carried out under light-protected conditions.

[0050] 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.

[0051] Example 1

[0052] according to Figure 1 The flowchart shown illustrates the preparation of compound P2T, as detailed below:

[0053] 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde (711 mg, 2.0 mmol), 1,2,6-trimethylpyridine iodide (249 mg, 1.0 mmol), and ethanol (20 mL) were mixed, and then 5 drops of piperidine were added dropwise. The mixture was refluxed (80 °C) under an argon atmosphere for 12 h. The resulting product system was cooled and filtered. The filter cake was dissolved in 20 mL of acetone, and an equal volume of saturated KPF6 aqueous solution was added. The resulting mixture was stirred at room temperature for 4 h. The organic phase of the resulting product system was removed by vacuum evaporation. The crude product was purified by silica gel column chromatography (the eluent used was dichloromethane:methanol = 20:1 by volume) to obtain compound P2T in 83% yield.

[0054] Figure 2 The image shows the 1H NMR spectrum of the compound P2T. Figure 3 The image shows the carbon NMR spectrum of the compound P2T. Figure 2 andFigure 3 The anionic group is not embodied in the structural formula; the results show that P2T synthesis is successful.

[0055] Test Example 1

[0056] This test example uses human umbilical vein endothelial cell extracellular vesicles for experiments, as follows:

[0057] Extracellular vesicle separation: human umbilical vein endothelial cells are cultured in vitro, and cell supernatant is collected; the cell supernatant is placed in a centrifuge and centrifuged at 500g for 10 min, and the precipitate is removed. Then the obtained supernatant is centrifuged at 2000g for 20 min to remove the precipitate, and finally the obtained supernatant is centrifuged at 5000g for 30 min to remove the precipitate. The obtained supernatant is passed through a 0.2μm filter, and then an ultracentrifuge is used to centrifuge at 130000g for 2h, the supernatant is discarded, the precipitate is resuspended with PBS, and then the ultracentrifuge is used again to centrifuge at 130000g for 2h, the supernatant is discarded, and the precipitate (mass 100μg) is resuspended with PBS (volume 100μL) to obtain an extracellular vesicle suspension.

[0058] Extracellular vesicle labeling: compound P2T (solid powder) is dissolved with dimethyl sulfoxide to prepare a P2T stock solution with a concentration of 5mmol / L; 0.8μL of the P2T stock solution (5mmol / L) is added to the extracellular vesicle (100μg) suspension (100μL), and PBS is used to make up to 1000μL, and thoroughly mixed by oscillation. Incubate at 37℃ for 2h in the dark; then the obtained mixture is placed in an ultracentrifuge and centrifuged at 130000g for 2h to remove the compound P2T that is not labeled in the supernatant, and resuspended with PBS to obtain a compound P2T-labeled extracellular vesicle suspension.

[0059] 1. Use a transmission electron microscope to observe the unlabeled extracellular vesicle sample (EVs) and the compound P2T-labeled extracellular vesicle sample (P2T-EVs). Figure 4 The transmission electron microscope images of the unlabeled extracellular vesicle sample and the compound P2T-labeled extracellular vesicle sample show that the extracellular vesicles are small vesicles with a bilayer structure with a particle size of about 100nm, and the compound P2T can be combined on the surface of the extracellular vesicle membrane.

[0060] 2. The surface of the extracellular vesicle membrane is negatively charged, and the compound P2T is positively charged. Use a nanoparticle size potential instrument to detect the ZETA potential of EVs and P2T-EVs. Figure 5The ZETA potential test result chart of the unmarked extracellular vesicle sample and the extracellular vesicle sample marked by the compound P2T shows that the potential of P2T-EVs is slightly higher than that of EVs, proving that the compound P2T is combined on the surface of the extracellular vesicle membrane through positive and negative charge adsorption.

[0061] 3. The particle size of the unmarked extracellular vesicle sample and the extracellular vesicle sample marked by the compound P2T is determined. Figure 6 The particle size test result chart of the unmarked extracellular vesicle sample and the extracellular vesicle sample marked by the compound P2T shows that the compound P2T in the present application does not affect the particle size of the extracellular vesicle.

[0062] 4. The extracellular vesicle labeling efficiency of the compound P2T is detected by a nano-flow experiment, and the commonly used commercial extracellular vesicle tracer probe PKH26 is used as a control. Figure 7 The extracellular vesicle labeling efficiency test result chart of the compound P2T and the probe PKH26 shows that the labeling efficiency of the compound P2T (79.9%) is higher than that of the probe PKH26 (39.4%) in labeling the extracellular vesicle.

[0063] 5. The effect of the compound P2T in tracing the extracellular vesicle is evaluated by a cell immunofluorescence staining experiment, and the commonly used commercial extracellular vesicle tracer probe PKH26 is used as a control. Specifically, the labeled P2T-EVs or PKH26-EVs are added to the culture medium of the neuron cell line HT22 for co-culture for 48 h, and then a cell immunofluorescence staining experiment is performed. The fluorescence signal is recorded by a laser confocal microscope on the 1st and 3rd day after staining, and the intensity of the fluorescence signal is analyzed by using software. Figure 8 The laser confocal microscope chart after the P2T-EVs and the PKH26-EVs are co-cultured with the neuron cell line HT22 and stained, Figure 9 The fluorescence signal intensity comparison chart shows that the fluorescence intensity of P2T-EVs is higher than that of PKH26-EVs.

[0064] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A fluorescent compound, characterized in that, It includes cationic and anionic groups, wherein the anionic group is selected from iodide ions, bromide ions, or PF6. - The cationic group has the structure shown in Formula I: Formula I.

2. The method for preparing the fluorescent compound according to claim 1, characterized in that, Includes the following steps: 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridine iodide, an acid-binding agent, and an organic solvent were mixed and subjected to a condensation reaction to obtain a fluorescent compound with an iodide ion as the anionic group. The fluorescent compound with iodide ion as the anionic group was mixed with an anionic group donor, an organic solvent, and water, and then subjected to a displacement treatment to obtain a compound with bromide ion or PF6 as the anionic group. - The fluorescent compound; wherein the anionic group donor is a bromide ion or PF6. - .

3. The preparation method according to claim 2, characterized in that, The acid-binding agent includes piperidine, sodium ethoxide, or sodium methoxide.

4. The preparation method according to claim 2 or 3, characterized in that, The temperature of the condensation reaction is the reflux temperature of the system, and the time of the condensation reaction is 10~15h.

5. The preparation method according to claim 2, characterized in that, The anionic group donor includes KPF6 or KBr; the temperature of the displacement treatment is 20~30℃, and the time of the displacement treatment is 3~5h.

6. The use of the fluorescent compound of claim 1 in the preparation of extracellular vesicle tracer reagents.

7. A method for labeling extracellular vesicles for non-disease diagnosis or treatment purposes, characterized in that, Includes the following steps: The probe, extracellular vesicles, and dispersant are mixed and incubated, wherein the probe is the fluorescent compound of claim 1.

8. The method according to claim 7, characterized in that, The extracellular vesicles are extracellular vesicles of human umbilical vein endothelial cells; the ratio of the probe to the extracellular vesicles is 4µmol:0.05~0.15g.

9. The method according to claim 7 or 8, characterized in that, The dispersant comprises an organic solvent and a buffer solution; the mixing comprises: first mixing the probe with the organic solvent to obtain a probe stock solution; second mixing the extracellular vesicles with a portion of the buffer solution to obtain an extracellular vesicle suspension; and third mixing the probe stock solution, the extracellular vesicle suspension, and the remaining buffer solution.

10. The method according to claim 7 or 8, characterized in that, The incubation temperature is 35~37℃, and the time is 1.5~2.5h; the incubation is carried out under light-protected conditions.

Images