Fluorescent compound, preparation method and application thereof, and method for marking extracellular vesicles

Abstract

The invention provides a fluorescent compound, a preparation method and application thereof, and a method for marking extracellular vesicles, and belongs to the technical field of biological medicines. The fluorescent compound provided by the invention comprises a cationic group and an anionic group, the anionic group is selected from iodide ions, bromide ions or PF6 <->, and the cationic group has a structure as shown in a formula I. The fluorescent compound provided by the invention has relatively high extracellular vesicle labeling efficiency, and the labeled extracellular vesicles have relatively high fluorescence intensity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a fluorescent compound, a preparation method and application thereof, and a method for labeling extracellular vesicles. Background Art

[0002] Before extracellular vesicle preparations are used in the clinical treatment of diseases, it is necessary to understand the biological behavior of extracellular vesicles after entering the human body in order to determine their safety. Therefore, mastering mature extracellular vesicle tracing technology is of great significance to promote the clinical transformation of extracellular vesicle treatment of diseases. At present, the most commonly used commercial extracellular vesicle tracing probes in academia are lipophilic fluorescent probes, such as PKH26. However, the extracellular vesicle labeling efficiency of this type of probe and the fluorescence intensity of the labeled extracellular vesicles still need to be improved. Summary of the invention

[0003] The purpose of the present invention is to provide a fluorescent compound and a preparation method and application thereof, and a method for labeling extracellular vesicles. The fluorescent compound provided by the present invention has a high extracellular vesicle labeling efficiency, and the extracellular vesicles labeled therewith have a high fluorescence intensity.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a fluorescent compound, comprising a cationic group and an anionic group, wherein the anionic group is selected from iodine ion, bromide ion or PF6 - , the cationic group has a structure shown in Formula I:

[0006]

[0007] Preferably, the anionic group comprises PF6 - , bromide ion or iodide ion.

[0008] The present invention provides a method for preparing the fluorescent compound described in the above technical solution, comprising the following steps:

[0009] 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridinium iodide, an acid-binding agent and an organic solvent are mixed to carry out a condensation reaction to obtain a fluorescent compound whose anion group is an iodide ion;

[0010] The fluorescent compound whose anion group is iodide ion is mixed with anion group donor, organic solvent and water, and then subjected to replacement treatment to obtain a fluorescent compound whose anion group is bromide ion or PF6 - wherein the anion group in the anion group donor is a bromide 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 to 15 hours.

[0013] Preferably, the anion group donor includes KPF6 or KBr; the temperature of the replacement treatment is 20 to 30°C, and the time of the replacement treatment is 3 to 5 hours.

[0014] The present invention provides the use of the fluorescent compound described in the above technical solution in preparing an extracellular vesicle tracing reagent.

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

[0016] The probe, the extracellular vesicles and the dispersant are mixed and incubated, wherein the probe is the fluorescent compound described in the above technical solution.

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

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

[0019] Preferably, the incubation temperature is 35-37° C., and the time is 1.5-2.5 h; and the incubation is carried out in a light-proof condition.

[0020] Beneficial effects: The fluorescent compound provided by the present invention (denoted as compound P2T) has a positive charge, and the surface of the extracellular vesicle membrane has a negative charge. Compound P2T is combined with the surface of the extracellular vesicle membrane through the adsorption of positive and negative charges, so compound P2T has a high extracellular vesicle labeling efficiency. At the same time, compound P2T provided by the present invention has aggregation-induced emission (AIE) properties, that is, compound P2T can emit strong fluorescence in an aggregated state, so the extracellular vesicles labeled by it have a high fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flow chart for preparing compound P2T;

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

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

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

[0025] Figure 5 The graph shows the ZETA potential test results of the unlabeled extracellular vesicle sample and the extracellular vesicle sample labeled with compound P2T;

[0026] Figure 6 The graphs are the particle size test results of the unlabeled extracellular vesicle sample and the extracellular vesicle sample labeled with compound P2T;

[0027] Figure 7 This is a graph showing the test results of the extracellular vesicle labeling efficiency of compound P2T and probe PKH26;

[0028] Figure 8 Laser confocal microscopy images of P2T-EVs and PKH26-EVs after co-culture and staining with the neuronal cell line HT22;

[0029] Fig. 9 The fluorescence signal intensity comparison chart is shown in Figure 2. DETAILED DESCRIPTION

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

[0031]

[0032] The anion group of the fluorescent compound in the present invention is selected from PF6 - , bromide ion or iodide ion; the use of the above anion groups is beneficial to reducing the toxicity of the fluorescent compound and improving the biocompatibility.

[0033] At present, the most commonly used commercial extracellular vesicle tracing probes in academia are lipophilic fluorescent probes, such as PKH26. However, the extracellular vesicle labeling efficiency of such probes and the fluorescence intensity of the labeled extracellular vesicles still need to be improved. Moreover, lipophilic fluorescent probes will have a strong quenching effect when they are aggregated, which will result in a relatively short time for tracing extracellular vesicles. In addition, such probes will affect the particle size of extracellular vesicles and thus affect the cell's uptake of extracellular vesicles and the half-life of extracellular vesicles in the body. Therefore, it is of great significance to develop extracellular vesicle tracing probes with excellent tracing ability.

[0034] Aggregation-induced emission (AIE) materials show great potential in the field of biological imaging. AIE fluorescent compounds and nanoparticles formed by them have the advantages of high brightness, low background noise, uniform photostability, good biocompatibility, excellent tissue penetration ability and higher spatial resolution. The 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, the present invention designs a fluorescent compound (i.e., compound P2T), which can be used as an AIE extracellular vesicle tracing probe, provides a new method for in vivo tracing of extracellular vesicles, and provides strong support for further clinical transformation research of extracellular vesicles. The test case results show that compared with the currently commonly used commercial extracellular vesicle tracing probe PKH26, the compound P2T in the present invention has a higher extracellular vesicle labeling efficiency. This is because the compound P2T has a positive charge, the surface of the extracellular vesicle membrane has a negative charge, and the compound P2T is combined with the surface of the extracellular vesicle membrane through the adsorption of positive and negative charges. At the same time, the extracellular vesicles labeled with compound P2T in the present invention have a higher fluorescence intensity, because compound P2T has aggregation-induced emission (AIE) properties, that is, compound P2T can emit strong fluorescence in an aggregated state, and the time of tracing extracellular vesicles is longer. In addition, compound P2T in the present invention does not affect the particle size of extracellular vesicles, and thus does not affect the cell's uptake of extracellular vesicles and the half-life of extracellular vesicles in vivo.

[0035] The present invention provides a method for preparing 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-trimethylpyridinium iodide, an acid-binding agent and an organic solvent are mixed to carry out a condensation reaction to obtain a fluorescent compound whose anion group is an iodide ion;

[0037] The fluorescent compound whose anion group is iodide ion is mixed with anion group donor, organic solvent and water, and then subjected to replacement treatment to obtain a fluorescent compound whose anion group is bromide ion or PF6 - wherein the anion group in the anion group donor is a bromide ion or PF6 - .

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

[0039] The present invention mixes 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridinium iodide, an acid binding agent and an organic solvent, performs a condensation reaction, and obtains a fluorescent compound whose anion group is an iodide ion. As an embodiment of the present invention, the molar ratio of the 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde to 1,2,6-trimethylpyridinium iodide can be 2:0.8-1.2, specifically 2:1. As an embodiment of the present invention, the acid binding agent can include piperidine, sodium ethoxide or sodium methoxide, and the organic solvent can be an alcohol solvent, specifically ethanol; the present invention has no special limitation on the amount of the acid binding agent and the organic solvent, as long as the reaction can be carried out smoothly.

[0040] As an embodiment of the present invention, the temperature of the condensation reaction can be the reflux temperature of the system. Taking ethanol as an organic solvent, the temperature of the condensation reaction can be 80°C; the time of the condensation reaction can be 10 to 15 hours, specifically 12 hours; the condensation reaction can be carried out in a protective atmosphere, specifically argon atmosphere. As an embodiment of the present invention, after the condensation reaction, it is preferred that the product system obtained after the condensation reaction is cooled and then filtered, and the obtained filter cake is a fluorescent compound whose anion group is iodide ion.

[0041] After obtaining the fluorescent compound whose anion group is iodide ion, the present invention mixes the fluorescent compound whose anion group is iodide ion with anion group donor, organic solvent and water, and performs substitution treatment to obtain a fluorescent compound whose anion group is bromide ion or PF6 - wherein the anion group in the anion group donor is a bromide ion or PF6 - . As an embodiment of the present invention, the anion group donor may include KPF6 or KBr; the organic solvent may be acetone, and the volume ratio of the organic solvent to water may be 1:0.8-1.2, specifically 1:1; the amount of the anion group donor is based on ensuring that it forms a saturated solution with water; the present invention has no special limitation on the specific amount of the organic solvent and water, as long as it can ensure that the replacement treatment proceeds smoothly. In the embodiment of the present invention, specifically, the fluorescent compound whose anion group is iodine ion is dissolved in an organic solvent, and then an equal volume of saturated anion group donor aqueous solution is added to carry out the replacement treatment.

[0042] As an embodiment of the present invention, the temperature of the replacement treatment can be 20-30°C, specifically room temperature (25°C); the time of the replacement treatment can be 3-5h, specifically 4h; the replacement treatment can be carried out under stirring. As an embodiment of the present invention, the replacement treatment preferably also includes: vacuum evaporating the mixture obtained after the replacement treatment to remove the organic phase, and then purifying the obtained crude product by silica gel column chromatography to obtain an anion group of bromide ion or PF6 - The fluorescent compound; the eluent used for the silica gel column chromatography purification may be dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be 20:1.

[0043] The present invention provides the use of the fluorescent compound described in the above technical solution in preparing an extracellular vesicle tracing reagent.

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

[0045] The probe, the extracellular vesicles and the dispersant are mixed and incubated, wherein the probe is the fluorescent compound described in the above technical solution.

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

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

[0048] As an embodiment of the present invention, the dispersant may include an organic solvent and a buffer solution, the organic solvent may specifically be dimethyl sulfoxide, and the buffer solution may specifically be PBS. As an embodiment of the present invention, the mixing may include: first mixing the probe with the organic solvent to obtain a probe mother solution; second mixing the extracellular vesicles with part of the buffer solution to obtain an extracellular vesicle suspension; and third mixing the probe mother solution, the extracellular vesicle suspension, and the remaining buffer solution. As an embodiment of the present invention, the concentration of the probe mother solution may be 4 to 6 mmol / L, specifically 5 mmol / L; the concentration of the extracellular vesicle suspension may be 0.5 to 2.0 g / L, specifically 1.0 g / L; the volume ratio of the probe mother 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 may be 35-37° C., and the incubation time may be 1.5-2.5 h, specifically 2 h; and the incubation is preferably carried out in a light-proof condition.

[0050] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Example 1

[0052] according to Figure 1 The compound P2T is prepared according to the flow chart shown in the figure, as follows:

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

[0054] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the compound P2T, Figure 3 is the carbon nuclear magnetic resonance spectrum of the compound P2T, Figure 2 and Figure 3 The structural formula does not reflect the anionic group; the results show that P2T was successfully synthesized.

[0055] Test Example 1

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

[0057] Isolation of extracellular vesicles: human umbilical vein endothelial cells were cultured in vitro and cell supernatants were collected; the cell supernatants were placed in a centrifuge and centrifuged at 500 g for 10 min to remove the precipitate, and then the supernatant was centrifuged at 2000 g for 20 min to remove the precipitate, and finally the supernatant was centrifuged at 5000 g for 30 min to remove the precipitate; the supernatant was passed through a 0.2 μm filter, and then centrifuged at 130,000 g for 2 h using an ultracentrifuge, the supernatant was discarded, the precipitate was resuspended with PBS, and then centrifuged at 130,000 g for 2 h using an ultracentrifuge again, the supernatant was discarded, and the precipitate (mass 100 μg) was resuspended with PBS (volume 100 μL) to obtain an extracellular vesicle suspension.

[0058] Labeling of extracellular vesicles: Compound P2T (solid powder) was dissolved in dimethyl sulfoxide to prepare a P2T mother solution with a concentration of 5 mmol / L; 0.8 μL of the P2T mother solution (5 mmol / L) was added to the extracellular vesicle (100 μg) suspension (100 μL), and the volume was adjusted to 1000 μL with PBS, and the mixture was thoroughly shaken and mixed, and incubated at 37°C in the dark for 2 h; the resulting mixture was then placed in an ultracentrifuge and centrifuged at 130,000 g for 2 h, the unlabeled compound P2T in the supernatant was removed, and the compound P2T was resuspended with PBS to obtain an extracellular vesicle suspension labeled with compound P2T.

[0059] 1. Use transmission electron microscopy to observe unlabeled extracellular vesicle samples (EVs) and extracellular vesicle samples labeled with compound P2T (P2T-EVs). Figure 4 The transmission electron micrographs of unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T show that the extracellular vesicles are bilayer vesicles with a particle size of about 100 nm, and compound P2T can bind to 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. The zeta potential of EVs and P2T-EVs was detected using a nanoparticle size potentiometer. Figure 5The figure shows the ZETA potential test results of unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T. The results show that the potential of P2T-EVs is slightly higher than that of EVs, proving that compound P2T is bound to the surface of the extracellular vesicle membrane through positive and negative charge adsorption.

[0061] 3. Measure the particle size of unlabeled extracellular vesicle samples and extracellular vesicle samples labeled with compound P2T. Figure 6 The graph shows the particle size test results of an unlabeled extracellular vesicle sample and an extracellular vesicle sample labeled with compound P2T. The results show that compound P2T in the present invention does not affect the particle size of extracellular vesicles.

[0062] 4. The extracellular vesicle labeling efficiency of compound P2T was detected by nanoflow cytometry, and the commonly used commercial extracellular vesicle tracer probe PKH26 was used as a control. Figure 7 This is a graph showing the test results of the extracellular vesicle labeling efficiency of compound P2T and probe PKH26. The results show that the efficiency of compound P2T labeling extracellular vesicles (79.9%) is higher than the efficiency of probe PKH26 labeling extracellular vesicles (39.4%).

[0063] 5. The effect of compound P2T on tracing extracellular vesicles was evaluated by cell immunofluorescence staining experiments, and the currently commonly used commercial extracellular vesicle tracing probe PKH26 was used as a control. Specifically, the labeled P2T-EVs or PKH26-EVs were added to the culture medium of the neuronal cell line HT22 and co-cultured for 48 hours, followed by cell immunofluorescence staining experiments. On the 1st and 3rd day after staining, the fluorescence signal was recorded using a laser confocal microscope and the intensity of the fluorescence signal was analyzed using software. Figure 8 This is a laser confocal microscopy image of P2T-EVs and PKH26-EVs co-cultured with the neuronal cell line HT22 and stained. Fig. 9 This is a comparison chart of fluorescence signal intensity; the results show that the fluorescence intensity of P2T-EVs is higher than that of PKH26-EVs.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

2. The method for preparing the fluorescent compound according to claim 1, characterized in that: The following steps are involved: 5-[4-(diphenyl-amino)phenyl]thiophene-2-acetaldehyde, 1,2,6-trimethylpyridinium iodide, an acid-binding agent and an organic solvent are mixed to carry out a condensation reaction to obtain a fluorescent compound whose anion group is an iodide ion; The fluorescent compound whose anion group is iodide ion is mixed with anion group donor, organic solvent and water, and then subjected to replacement treatment to obtain a fluorescent compound whose anion group is bromide ion or PF6 - wherein the anion group in the anion 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 to 15 hours.

5. The preparation method according to claim 2, characterized in that: The anion group donor includes KPF6 or KBr; the temperature of the replacement treatment is 20-30°C, and the time of the replacement treatment is 3-5h.

6. Use of the fluorescent compound according to claim 1 in preparing an extracellular vesicle tracing reagent.

7. A method for labeling extracellular vesicles, characterized in that: The following steps are involved: The probe, the extracellular vesicles and the dispersant are mixed and incubated, wherein the probe is the fluorescent compound according to claim 1.

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

9. The method according to claim 7 or 8, characterized in that: The dispersant includes an organic solvent and a buffer solution; the mixing includes: first mixing the probe with the organic solvent to obtain a probe mother solution; second mixing the extracellular vesicles with part of the buffer solution to obtain an extracellular vesicle suspension; and third mixing the probe mother 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° C., and the time is 1.5-2.5 h; the incubation is carried out in a light-proof condition.

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