JP7423888B2 - Dye for staining lipid bilayer membranes and method for staining lipid bilayer membranes using the same - Google Patents

Description

The present invention relates to a novel dye for staining lipid bilayer membranes and a method for staining lipid bilayer membranes using the same.

In 1983, it was discovered that vesicles with a diameter of about 100 nm made of a lipid bilayer membrane were secreted from reticulocytes, and were named exosomes (see Non-Patent Document 1). Around the time of the discovery of exosomes, it was discovered that various cells secrete membrane vesicles of different sizes. The International Society for Extracellular Vesicles (ISEV) recommends the use of extracellular vesicles as a general term for vesicles secreted from these cells. It is becoming clear that extracellular vesicles, including exosomes, transport various physiologically active substances while moving between cells. In multicellular organisms, interactions between cells are involved in a variety of life activities, and their breakdown can lead to various diseases. It is expected that this research will lead to an understanding of the molecular mechanisms underlying cancer, the pathophysiology of various diseases, and the development of new diagnostic and therapeutic methods.

In order to study the formation and secretion pathway of extracellular vesicles including exosomes, dyes for staining the lipid bilayer membrane of extracellular vesicles are required. As conventionally used dyes for fluorescent staining of exosomes, for example, those represented by the following formula are known (see Patent Documents 1 and 2).

US Patent No. 5,665,328 US Patent No. 8,894,976

Johnstone, R. M., Adam, M., Hammond, J. R. et al.: Vesicle formation during reticulocyte maturation. Association of membrane plasma activities with released vesicles (exosomes). J. Biol. Chem., 262, 9412-9420 (1987)

However, it has been pointed out that when the fluorescent dye represented by the above formula is used to stain the membranes of exosomes, it may form particles, enlarge the exosomes, and change their properties. At the same time, it has been pointed out that the particles migrate inside the vesicles and cause background light emission, resulting in a decrease in sensitivity.

The present invention was developed in view of the above circumstances, and has a high retention property in lipid bilayer membranes, and even when used for staining extracellular vesicles, it can perform fluorescence measurements with high sensitivity without changing the particle size etc. The object of the present invention is to provide a dye for staining lipid bilayer membranes and a method for staining lipid bilayer membranes using the same.

A first aspect of the present invention in accordance with the above object is to solve the above problems by providing a dye for staining lipid bilayer membranes represented by the following general formula (I).

In the above general formula (I),
X is oxygen, sulfur, selenium, or an atom or atomic group represented by the formula -CR ¹¹ ₂ - (R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms),
n is a natural number of 1 or 2,
R ¹ is an alkylene group having 1 to 12 carbon atoms,
R ² is an atomic group containing a polyoxyethylene group represented by the formula (CH ₂ CH ₂ O) _m (m is a natural number from 1 to 10),
R ³ is an alkyl group having 6 to 20 carbon atoms,
L ¹ , L ² and L ³ each independently represent an ester bond (-CO-O-), an amide bond (-CO-NH-), a urethane bond (-NH-CO-O-), and a urea bond ( -NH-CO-NH-) is a bonding group selected from the group consisting of
q is a natural number from 1 to 5.

A second aspect of the present invention includes a step of preparing a sample solution containing a lipid bilayer membrane, and a dye for staining a lipid bilayer membrane represented by the above general formula (I) and/or a composition for staining containing the same. Provided is a method for staining a lipid bilayer membrane, which includes a step of preparing a lipid bilayer membrane, and a step of adding the lipid bilayer membrane staining dye or the staining composition to the sample solution to stain the lipid bilayer membrane. This solves the above problem.

In the lipid bilayer membrane staining dye according to the first aspect of the present invention and the lipid bilayer membrane staining method according to the second aspect of the present invention, the lipid bilayer membrane is a cell membrane or a lipid bilayer derived from a cell membrane. It may also be a molecular membrane.

In the lipid bilayer membrane staining dye according to the first aspect of the present invention and the method for staining a lipid bilayer membrane according to the second aspect of the present invention, the lipid bilayer membrane may be an exosome membrane.

In the dye for staining lipid bilayer membranes according to the first aspect of the present invention and the method for staining lipid bilayer membranes according to the second aspect of the present invention, X in the general formula (I) is oxygen or the formula - It may also be an atomic group represented by C(CH ₃ ) ₂ -.

In the dye for staining lipid bilayer membranes according to the first aspect of the present invention and the method for staining lipid bilayer membranes according to the second aspect of the present invention, L ¹ , L ² and L in the above general formula (I) ³ may be an amide bond.

In the lipid bilayer membrane staining dye according to the first aspect of the present invention and the lipid bilayer membrane staining method according to the second aspect of the present invention, the lipid bilayer membrane staining dye has the following formula 9, It may be represented by either one of 15 and 20.

According to the present invention, a dye for staining lipid bilayer membranes and a method for staining lipid bilayer membranes, which have high retention properties in lipid bilayer membranes and can be suitably used for staining cell membranes and extracellular vesicles such as exosomes, are provided. provided. Furthermore, the dye for staining lipid bilayer membranes provided by the present invention does not easily form particles or aggregates under staining conditions, so it can be used to stain small extracellular vesicles such as exosomes. Enables highly accurate and sensitive fluorescence measurements without changing size or creating artifacts.

1 is a fluorescence micrograph showing the relationship between the amount of exosomes present and an image stained with the dye for staining lipid bilayer membranes of the present invention. It is a fluorescence micrograph which shows the relationship with the stained image with the dye for lipid bilayer membrane staining of this invention in the presence/absence of exosome. 1 is a fluorescence micrograph showing an image stained with a conventional lipid bilayer membrane staining dye in the presence of exosomes. It is a graph showing the results of nanoparticle tracking analysis when exosomes are stained with a conventional dye for staining lipid bilayer membranes. It is a graph showing the results of nanoparticle tracking analysis when exosomes are stained with the dye for lipid bilayer membrane staining of the present invention. 1 is a differential interference micrograph and a fluorescence micrograph showing the results of a HeLa cell staining test using a conventional lipid bilayer membrane staining dye and a lipid bilayer membrane staining dye of the present invention. 1 is a differential interference micrograph and a fluorescence micrograph showing the results of a HeLa cell staining test using a conventional lipid bilayer membrane staining dye and a lipid bilayer membrane staining dye of the present invention.

Next, embodiments embodying the present invention will be described to provide an understanding of the present invention.

The dye for staining lipid bilayer membranes (hereinafter sometimes abbreviated as "dye for staining lipid bilayer membranes") according to the first embodiment of the present invention is represented by the following general formula (I). .

^In _the ^above general formula (I), It is preferably oxygen or an atomic group represented by the formula -C(CH ₃ ) ₂ -.

In the above general formula (I), n is a natural number of 1 or 2.

In the above general formula (I), R ¹ is an alkylene group having 1 to 12 carbon atoms, preferably 2 to 8 carbon atoms, and more preferably 4 to 6 carbon atoms.

In the above general formula (I), R ² is represented by the formula (CH ₂ CH ₂ O) _m (m is a natural number of 1 to 10, preferably 2 to 8, more preferably 3 to 5). It is an atomic group containing a polyoxyethylene group. The presence of polyoxyethylene groups gives the dye for lipid bilayer membrane staining water solubility, eliminating the need for organic solvents in preparing the staining solution and suppressing the formation of aggregates and particles in a hydrophilic environment. .

In the above general formula (I), R ³ is an alkyl group having 6 to 20 carbon atoms, preferably 8 to 18 carbon atoms, more preferably 10 to 16 carbon atoms, and preferably a straight-chain alkyl group.

In the above general formula (I), L ¹ , L ² and L ³ each independently represent an ester bond (-CO-O-), an amide bond (-CO-NH-), a urethane bond (-NH-CO -O-) and a urea bond (-NH-CO-NH-), preferably an amide bond.

The dye for lipid bilayer membrane staining has a carboxylic acid group in the side chain, which suppresses the permeability of the lipid bilayer membrane and allows it to be used for staining lipid bilayer membranes inside cells and extracellular vesicles to be stained. Transfer of pigment can be suppressed. In the above general formula (I), q is a natural number of 1 to 5, preferably 1 or 2.

Since the lipid bilayer membrane staining dye represented by the above general formula (I) has a monovalent positive charge, it forms a salt with a counter anion. The counter anion may be any anion as long as it does not inhibit fluorescence or have cytotoxicity. Specific examples of counteranions include chloride ion, bromide ion, perchlorate ion, nitrate ion, acetate ion, trifluoroacetate ion, and the like.

Preferred specific examples of dyes for staining lipid bilayer membranes include those represented by the following formulas 9, 15, and 20.

The dyes for staining lipid bilayer membranes represented by formulas 9, 15, and 20 above can be synthesized using any known method, and specific examples of their synthetic routes are shown in Examples below. Examples include those shown below.

The method for staining a lipid bilayer membrane according to the second embodiment of the present invention (hereinafter may be abbreviated as "method for staining a lipid bilayer membrane") is a method for staining a lipid bilayer membrane using a sample solution containing a lipid bilayer membrane. a step of preparing the lipid bilayer membrane staining dye and/or a staining composition containing the same according to the first embodiment of the present invention; and a step of preparing the lipid bilayer membrane staining dye or the staining composition according to the first embodiment of the present invention. The method includes the step of adding a substance to a sample solution and staining the lipid bilayer membrane.

The lipid bilayer membrane to be stained may contain amphiphilic lipids such as phospholipids, glycolipids, and sterols, and may be of any type as long as it can be stained with a dye for staining lipid bilayer membranes. It may also contain proteins and the like. Examples of lipid bilayer membranes to be stained include cell membranes of animal cells and extracellular vesicles such as exosomes having lipid bilayer membranes derived therefrom.

A sample solution containing a staining target such as those described above is prepared by dispersing the staining target in an appropriate solution. When the object to be stained is a cell, the solution may contain a buffer, a medium, and the like. In the case of extracellular vesicles such as exosomes, further treatment such as isolation by ultracentrifugation may be performed.

Since the dye for staining lipid bilayer membranes according to the first embodiment of the present invention is water-soluble, staining can be performed by directly adding it to a sample solution. A dyeing composition containing a dye for staining lipid bilayer membranes may be prepared by dissolving the dye for dyeing in a suitable solvent. As described above, since the dye for staining lipid bilayer membranes according to the first embodiment of the present invention is water-soluble, the solvent does not need to contain an organic solvent.

Staining is performed by adding a lipid bilayer membrane staining dye or a staining composition containing the same to the sample solution. If necessary, treatments such as incubation for a predetermined period of time can also be performed as appropriate. Observation of the stained image can be performed using any known method such as a fluorescence microscope. A method such as nanoparticle tracking analysis (NTA) can be used to analyze the number and size of particles such as exosomes.

Next, examples performed to confirm the effects of the present invention will be described. In addition, in the following description, "intermediate represented by formula n" and "dye for lipid bilayer membrane staining represented by formula n" are respectively referred to as "intermediate n" and "dye for lipid bilayer membrane staining". It may be abbreviated as "dye n".
Example 1: Synthesis of synthetic intermediate 1 of dye for lipid bilayer membrane staining

4,7,10-trioxa-1,13-tridecanediamine was added to the eggplant flask. Chloroform was added to dissolve, and Boc ₂ O/chloroform solution was added dropwise. After stirring at room temperature, the mixture was separated into layers using saturated aqueous sodium bicarbonate solution/chloroform, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain Intermediate 1.

Synthesis of intermediate 2

Palmitic acid and thionyl chloride were added to an eggplant flask, connected to a calcium chloride tube, and stirred. One drop of DMF was added and stirred at 40°C for 1 hour. After concentration under reduced pressure, anhydrous toluene was added and the mixture was concentrated under reduced pressure again to obtain palmitoyl chloride. L-glutamic acid-γ-t-butyl ester, THF, water, and diisopropylethylamine were added to an eggplant flask and stirred. Palmitoyl chloride was dissolved in THF and added to an eggplant flask. The layers were separated using chloroform/5% citric acid aqueous solution, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain Intermediate 2.

Synthesis of intermediate 3

Chloroform, Intermediate 2, and diisopropylethylamine were added to an eggplant flask, and a calcium chloride tube was connected to the flask, followed by stirring. DSC (di(N-succinimidyl) carbonate)/chloroform solution was added. After stirring at room temperature, the mixture was separated with chloroform/water, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (hexane/ethyl acetate) to obtain intermediate 3.

Synthesis of intermediate 4

2,3,3-trimethylindolenine and acetonitrile were added to an eggplant flask and stirred. Bromohexanoic acid was added, a reflux tube was connected, and the mixture was heated under reflux overnight. The mixture was cooled to room temperature, and 4M HCl/THF and ether were added and collected by filtration. Intermediate 4 was obtained by suspending the crude product in ether and filtering it again.

Synthesis of intermediate 5

Intermediate 4 and pyridine were added to an eggplant flask, connected to a reflux tube, and stirred. Trimethyl orthoformate was added and the mixture was heated under reflux overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/1M hydrochloric acid, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain Intermediate 5.

Synthesis of intermediate 6

Intermediate 5, DMF, and HATU were added to an eggplant flask, connected to a calcium chloride tube, and stirred. A DMF solution containing Intermediate 1 and diisopropylethylamine was added and stirred overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/5% citric acid aqueous solution, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified on a silica gel column (chloroform/methanol).

Synthesis of intermediates 7 and 8

Intermediate 6 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. Intermediate 7 was obtained by concentration under reduced pressure. Intermediate 7, DMF, and diisopropylethylamine were added to an eggplant flask and stirred. A calcium chloride tube was connected, a DMF solution of Intermediate 3 was added, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, the layers were separated with chloroform/water, and the organic phase was washed with saturated brine and concentrated under reduced pressure. Purification was performed using a silica gel column (chloroform/methanol) to obtain Intermediate 8.

Synthesis of dye 9 for lipid bilayer membrane staining

Intermediate 8 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. It was concentrated under reduced pressure. The crude product was purified by reverse phase HPLC (water containing 0.1% TFA/acetonitrile) to obtain dye 9 for staining lipid bilayer membranes that emits red light.

Synthesis of intermediate 10

2-methylbenzoxazole and bromohexanoic acid were added to an eggplant flask, a reflux tube was connected, and the flask was heated under reflux overnight. It was cooled to room temperature and collected by filtration. Intermediate 10 was obtained by suspending the crude product in ether and filtering it again.

Synthesis of intermediate 11

Intermediate 10 and pyridine were added to an eggplant flask, connected to a reflux tube, and stirred. Trimethyl orthoformate was added and the mixture was heated under reflux overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/1M hydrochloric acid, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain Intermediate 11.

Synthesis of intermediate 12

Intermediate 11, DMF, and HATU were added to an eggplant flask, connected to a calcium chloride tube, and stirred. A DMF solution containing Intermediate 1 and diisopropylethylamine was added and stirred overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/5% citric acid aqueous solution, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain intermediate 12.

Synthesis of intermediates 13 and 14

Intermediate 12 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. Intermediate 13 was obtained by concentration under reduced pressure. Intermediate 13, DMF, and diisopropylethylamine were added to an eggplant flask and stirred. A calcium chloride tube was connected, a DMF solution of Intermediate 3 was added, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, the layers were separated with chloroform/water, and the organic phase was washed with saturated brine and concentrated under reduced pressure. Purification was performed using a silica gel column (chloroform/methanol) to obtain Intermediate 14.

Synthesis of dye 15 for lipid bilayer membrane staining

Intermediate 14 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. It was concentrated under reduced pressure. The crude product was purified by reverse phase HPLC (water containing 0.1% TFA/acetonitrile) to obtain dye 15 for staining lipid bilayer membranes that emitted green light.

Synthesis of intermediate 16

Intermediate 4 and pyridine were added to an eggplant flask, connected to a reflux tube, and stirred. Malonaldehyde dianilide hydrochloride was added and the mixture was heated under reflux overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/1M hydrochloric acid, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain intermediate 16.

Synthesis of intermediate 17

Intermediate 16, DMF, and HATU were added to an eggplant flask, connected to a calcium chloride tube, and stirred. A DMF solution containing Intermediate 1 and diisopropylethylamine was added and stirred overnight. The mixture was concentrated under reduced pressure, separated into layers with chloroform/5% citric acid aqueous solution, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The crude product was purified with a silica gel column (chloroform/methanol) to obtain intermediate 17.

Synthesis of intermediates 18, 19

Intermediate 17 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. Intermediate 18 was obtained by concentration under reduced pressure. Intermediate 18, DMF, and diisopropylethylamine were added to an eggplant flask and stirred. A calcium chloride tube was connected, a DMF solution of Intermediate 3 was added, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, the layers were separated with chloroform/water, and the organic phase was washed with saturated brine and concentrated under reduced pressure. Purification was performed using a silica gel column (chloroform/methanol) to obtain Intermediate 19.

Synthesis of dye 20 for lipid bilayer membrane staining

Intermediate 19 and chloroform were added to an eggplant flask and dissolved. TFA (trifluoroacetic acid) was added and stirred at 40°C for 1 hour. It was concentrated under reduced pressure. The crude product was purified by reverse phase HPLC (water containing 0.1% TFA/acetonitrile) to obtain dye 20 for staining lipid bilayer membranes that emitted purple light.

Example 2: Exosome staining test HEK293S, which secretes about 10 times more exosomes than normal cells, was cultured, and exosomes were separated by ultracentrifugation. Exosomes were dispersed in 100 μL of phosphate buffered saline (PBS) to prepare exosome-containing sample solutions with protein contents of 5 μg and 10 μg. Dyes 9, 15, and 20 for staining lipid bilayer membranes were added to this at a final concentration of 10 μmol/L, and the mixture was incubated at 37° C. for 30 minutes. After removing the lipid bilayer membrane staining dye remaining in the solution using an ultrafiltration membrane, the exosomes on the filtration membrane were collected and observed under a fluorescence microscope.

The results are shown in Figure 1. It was confirmed that with any dye for staining lipid bilayer membranes, the stained image was observed in proportion to the amount of exosomes.

Example 3: Confirmation of presence or absence of aggregate or particle formation Dyes 9, 15, and 20 for staining lipid bilayer membranes were added to an exosome-containing sample solution with a protein content of 10 μg prepared using the same method as in Example 2. Add so that the concentration is 10 μmol/L, or for comparison, add commercially available dyes for staining lipid bilayer membranes (PKH67, PKH26 (manufactured by Sigma-Aldrich)) so that the final concentration is 10 μmol/L. and incubated at 37°C for 24 hours. After removing the lipid bilayer membrane staining dye remaining in the solution using an ultrafiltration membrane, the exosomes on the filtration membrane were collected and observed under a fluorescence microscope. In addition, similar operations were performed in the absence of exosomes.

The results are shown in FIGS. 2 and 3. As shown in FIG. 2, with dyes 9, 15, and 20 for staining lipid bilayer membranes, staining images were observed in the presence of exosomes (Exo+), but not in the absence of exosomes (Exo-). On the other hand, as shown in FIG. 3, when staining was performed using PKH67 and PKH26 in the presence of exosomes (Exo+), irregular bright spots larger than the size of exosomes were observed. These results indicate that in PKH67 and PKH26, dye aggregates or particles were formed in addition to the exosome staining image, and bright spots derived from them were observed, whereas in the case of lipid bilayer membrane staining dye 9, In samples No. 15 and 20, no bright spots originating from aggregates or particles were observed.

4 and 5 show the results of measuring the particle number and particle diameter of each sample by nanoparticle tracking analysis (NTA). In FIGS. 4 and 5, "Exo" indicates the measurement results of an unstained exosome-containing sample solution. From FIG. 4, when staining was performed using PKH67 and PKH26, a decrease in the number of particles and an increase in the particle size were observed. This result indicates that PKH67 and PKH26 change the properties of exosomes.

On the other hand, the results in Figure 5 show that dyes 9, 15, and 20 for staining lipid bilayer membranes do not change the particle number or particle diameter, and these dyes for staining lipid bilayer membranes do not affect exosomes. This shows that it does not change the properties of

Example 5: HeLa cell staining test HeLa cells were seeded on a microwell slide (0.75×10 ⁴ cells/well). MEM medium (containing 10% FBS (fetal bovine serum)) was used as the medium, and after culturing overnight at 37° C. in a CO ₂ incubator, the cells were washed with MEM medium (containing or not containing FBS). MEM (0 or 10% FBS) solution (1 μmol/L) was prepared and added to the cells. After being allowed to stand at room temperature for 5 minutes, it was washed with MEM medium containing the corresponding FBS content. Observation was made using a fluorescence microscope (magnification x63).

The results are shown in FIG. 6 (when FBS-free MEM medium was used as the washing and staining solution) and FIG. 7 (when FBS-containing MEM medium was used as the washing and staining solution). In FIGS. 6 and 7, "FLU" indicates a fluorescent staining image, and "merged with DIC" indicates an image in which the fluorescent staining image is superimposed on the differential interference image. It was confirmed that in the absence of FBS in FIG. 6, lipid bilayer membrane staining dye 9 and PKH26 selectively and clearly stain cell membranes. In the case of PKH26, some migration into the cytoplasm and bright spots were observed. On the other hand, when Dil was used, no staining of cells was observed, and the presence of bright spots, which seemed to be caused by the formation of aggregates or particles, was also observed. In the presence of FBS in FIG. 7, a stained image in which the cell membrane was clearly stained only with dye 9 for staining lipid bilayer membranes was observed. In the case of Dil, bright spots, which are considered to be caused by the formation of aggregates or particles, were observed in the same manner as in the absence of FBS, and in the case of PKH26, similar bright spots were observed in the presence of FBS. Since Dil has low water solubility and aggregates into a suspended state in an aqueous solution, it is considered that it cannot stain cells. Although PKH26 is capable of staining cell membranes in the absence of FBS, it is thought that the staining image of cell membranes was not observed in the presence of FBS because the dye was adsorbed to the fat-soluble components in FBS. In contrast, dye 9 for staining lipid bilayer membranes is highly water-soluble and has high retention and localization in cell membranes, so it was confirmed that it selectively stains cell membranes regardless of the presence or absence of FBS. .

Claims (12)

A dye for staining lipid bilayer membranes represented by the following general formula (I).

In the above general formula (I),
X is oxygen, sulfur, selenium, or an atom or atomic group represented by the formula -CR ¹¹ ₂ - (R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms),
n is a natural number of 1 or 2,
R ¹ is an alkylene group having 1 to 12 carbon atoms,
R ² is an atomic group containing a polyoxyethylene group represented by the formula (CH ₂ CH ₂ O) _m (m is a natural number from 1 to 10),
R ³ is an alkyl group having 6 to 20 carbon atoms,
L ¹ , L ² and L ³ each independently represent an ester bond (-CO-O-), an amide bond (-CO-NH-), a urethane bond (-NH-CO-O-), and a urea bond ( -NH-CO-NH-) is a bonding group selected from the group consisting of
q is a natural number from 1 to 5. The dye for staining lipid bilayer membranes according to claim 1, wherein the lipid bilayer membrane is a cell membrane or a lipid bilayer membrane derived from a cell membrane. The dye for staining lipid bilayer membranes according to claim 2, wherein the lipid bilayer membrane is an extracellular vesicle membrane or an exosome membrane. The lipid compound according to any one of claims 1 to 3, wherein X in the general formula (I) is oxygen or an atomic group represented by the formula -C(CH ₃ ) ₂ -. Dye for molecular membrane staining. The dye for staining lipid bilayer membranes according to any one of claims 1 to 4, wherein L ¹ , L ² and L ³ in the general formula (I) are amide bonds. The dye for lipid bilayer membrane staining according to any one of claims 1 to 5, characterized in that it is represented by any one of the following formulas 9, 15, and 20.

preparing a sample solution containing a lipid bilayer membrane;
A step of preparing a lipid bilayer membrane staining dye represented by the following general formula (I) and/or a staining composition containing the same;
A method for staining a lipid bilayer membrane, comprising the step of adding the lipid bilayer membrane staining dye or the staining composition to the sample solution and staining the lipid bilayer membrane.

In the above general formula (I),
X is oxygen, sulfur, selenium, or an atom or atomic group represented by the formula -CR ¹¹ ₂ - (R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms),
n is a natural number of 1 or 2,
R ¹ is an alkylene group having 1 to 12 carbon atoms,
R ² is an atomic group containing a polyoxyethylene group represented by the formula (CH ₂ CH ₂ O) _m (m is a natural number from 1 to 10),
R ³ is an alkyl group having 6 to 20 carbon atoms,
L ¹ , L ² and L ³ each independently represent an ester bond (-CO-O-), an amide bond (-CO-NH-), a urethane bond (-NH-CO-O-), and a urea bond ( -NH-CO-NH-) is a bonding group selected from the group consisting of
q is a natural number from 1 to 5. 8. The method for staining a lipid bilayer membrane according to claim 7, wherein the lipid bilayer membrane is a cell membrane or a lipid bilayer membrane derived from a cell membrane. 9. The method for staining a lipid bilayer membrane according to claim 8, wherein the lipid bilayer membrane is an extracellular vesicle membrane or an exosome membrane. The lipid compound according to any one of claims 7 to 9, wherein X in the general formula (I) is oxygen or an atomic group represented by the formula -C(CH ₃ ) ₂ -. Method for staining molecular membranes. The method for staining a lipid bilayer membrane according to any one of claims 7 to 10, wherein L ¹ , L ² and L ³ in the general formula (I) are amide bonds. The lipid bilayer membrane staining dye according to any one of claims 7 to 11, wherein the lipid bilayer membrane staining dye is represented by any one of the following formulas 9, 15, and 20. Method for staining molecular membranes.