JP2011255319A - Method for producing vesicles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing vesicles, the method including simple production steps and providing vesicles of high encapsulation rate.SOLUTION: A double tube 2 including an outer tube 2a and an inner tube 2b which is arranged coaxially inside the outer tube 2a is used. A phospholipid is dissolved in an organic solvent soluble in water and having boiling point of 110°C or higher and polarity to obtain a phospholipid solution 10. The phospholipid solution 10 is ejected from the outer tube 2a while ejecting a first solution 11 containing water from the inner tube 2b. An electrode is opposed to the double tube 2 and voltage is applied to the outer tube 2a so that an electric field is formed between the double tube 2 and the electrode, and the phospholipid solution 10 ejected from the outer tube 2a is polarized to form fine droplets. Then the ejected fine droplets are brought in contact with a second solution containing water to dissolve the organic solvent of the fine droplets so that the phospholipid is brought in contact with the second solution to form a bilayer phospholipid membrane. As a result, vesicles of a microscale enclosing the first solution are produced.

Description

  The present invention relates to a method for producing microscale vesicles.

  Vesicles (also referred to as liposomes) are closed vesicles with an aqueous phase formed by a phospholipid bilayer membrane, and have been conventionally used as a biological membrane model for research. In addition, since vesicles have high biocompatibility, it is possible to enclose drugs and the like in the inner aqueous phase and administer them to the living body, and their use in various fields such as medical care and cosmetics has been studied.

  As a conventional vesicle production method, a hydration method is well known (see Non-Patent Document 1). This method uses a solution of phospholipid dissolved in chloroform / methanol to form a phospholipid thin film on the glass surface, and an aqueous solution is added to this to stir to produce a vesicle that encloses the aqueous solution. Is.

“New Development of Liposome Application-Toward Development of Artificial Cells” Kazunari Akiyoshi, Satoshi Sakurai, Naoto Oku, Ryoichi Kuboi (NTS, 2005/6)

  However, the conventional vesicle manufacturing method described above requires complicated operations. Further, since an extra aqueous solution is required at the time of production, there is also a problem that the substance encapsulation rate is low.

  Therefore, an object of the present invention is to provide a vesicle manufacturing method having a simple manufacturing process and a high encapsulation rate.

Means for Solving the Problems and Effects of the Invention

  The vesicle manufacturing method of the first invention uses a double pipe having an outer pipe and an inner pipe arranged coaxially in the outer pipe, has a boiling point of 110 ° C. or higher at atmospheric pressure, and has polarity. A step of extruding a phospholipid solution in which a phospholipid is dissolved in an organic compound solvent soluble in water from the outer tube and simultaneously extruding a first liquid containing water from the inner tube; and the double tube The phospholipid solution pushed out of the outer tube is polarized by applying an voltage to the outer tube and forming an electric field between the double tube and the electrode. A step of ejecting microdroplets composed of the first liquid and the phospholipid solution, and bringing the ejected microdroplets into contact with a second liquid containing water, so that the microdroplets By dissolving the organic compound solvent in the second liquid, the phosphorus And the quality is brought into contact with the second liquid to form a phospholipid bilayer membrane, and having a step of producing the vesicle microscale enclosing said first fluid.

  Since the organic compound solvent of the phospholipid solution has polarity, the organic compound solvent in the phospholipid solution pushed out from the outer tube is polarized by the influence of the electric field formed between the double tube and the electrode. As a result, a conical meniscus whose surface is electrically charged is formed at the tip of the double tube. When the electrostatic repulsion force exceeds the surface tension, the tip of the meniscus is separated into double-structured microdroplets in which the first liquid is covered with the phospholipid solution. The micro droplets are ejected in a spray form by electrostatic repulsion between the droplets. Then, when the ejected microdroplet comes into contact with the second liquid and the organic compound solvent is dissolved in water, the phospholipid and water come into contact with each other, thereby forming a phospholipid bilayer membrane. Thereby, a microscale vesicle enclosing the first liquid is manufactured.

According to this vesicle manufacturing method, a vesicle can be manufactured by a simple operation of pushing out a phospholipid solution and a first liquid from an outer tube and an inner tube while applying a voltage to the outer tube. Furthermore, since the vesicles can be continuously produced by continuously extruding from the outer tube and the inner tube at a constant flow rate, the productivity is excellent.
Further, since the first liquid pushed out from the inner pipe of the double pipe is included in the fine droplets, the first liquid is hardly wasted and can be enclosed in the vesicle at a high encapsulation rate.
In addition, if an organic compound solvent that volatilizes at room temperature is used as a solvent for dissolving phospholipids, when the vesicle is continuously produced, the solvent is volatilized and solidified at the tip of the double tube. It becomes difficult to eject a double droplet having a double structure. On the other hand, in the present invention, since a non-volatile organic compound solvent is used at room temperature, the vesicle can be continuously and stably manufactured without adhering to the tip of the double tube.

  The vesicle production method of the second invention is characterized in that, in the first invention, the organic compound solvent is polyethylene glycol.

  According to this configuration, since polyethylene glycol is an almost harmless organic compound, it is possible to produce a vesicle with high safety for a living body.

  In the vesicle production method of the third invention, in the second invention, the molecular weight of polyethylene glycol constituting the organic compound solvent is 100 to 600.

  According to this structure, since polyethylene glycol having a molecular weight of 100 to 600 is easily soluble in phospholipid, vesicles can be produced more reliably.

  A vesicle manufacturing method according to a fourth invention is characterized in that, in any one of the first to third inventions, the first liquid is an aqueous electrolyte solution or an aqueous solution of a solute having polarity.

  According to this configuration, electric charge collects in the first liquid pushed out from the inner tube due to the influence of the electric field, so that the polarization of the organic compound solvent in the phospholipid solution pushed out from the outer tube becomes larger. Therefore, since the electrostatic force on the surface of the meniscus becomes larger, the vesicle can be manufactured more reliably and a vesicle having a smaller diameter can be manufactured.

  In a vesicle manufacturing method according to a fifth aspect of the present invention, in the fourth aspect, the aqueous electrolyte solution constituting the first liquid is a polymer electrolyte aqueous solution.

  According to this configuration, the polyelectrolyte dissociates into a polyion having a positive or negative charge and a large number of low-molecular ions having a charge opposite to that of the polyion, and is more susceptible to an electric field. It becomes possible to carry out stably.

  The vesicle manufacturing method according to a sixth aspect of the present invention is the method for producing a vesicle according to any one of the first to fifth aspects, wherein a predetermined substance is added in advance to the first liquid pushed out from the inner tube. A vesicle containing the predetermined substance is manufactured.

  According to this configuration, a vesicle containing a predetermined substance can be easily manufactured.

  In the vesicle manufacturing method of the seventh invention, in the sixth invention, the predetermined substance is a cell, a drug, an enzyme, a functional food, a fragrance, an insecticide, an attractant, a biocatalyst, a protein, or a nucleic acid. It is characterized by including at least one.

It is the schematic of the vesicle manufacturing apparatus for performing the vesicle manufacturing method which concerns on embodiment of this invention. It is a principal part enlarged view of FIG. It is a microscope picture of the vesicle of Example 1, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 2, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 3, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 4, Comprising: (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 5, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 6, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 7, Comprising: (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 8, (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is the microscope picture of the vesicle of Example 9, Comprising: (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 10, Comprising: (a) is a photograph which shows a phospholipid bilayer membrane, (b) is a photograph which shows an internal aqueous phase. It is a microscope picture of the vesicle of Example 11, Comprising: It is a photograph which shows a phospholipid bilayer membrane.

Hereinafter, embodiments of the present invention will be described.
FIG. 1 is a diagram showing an outline of a vesicle manufacturing apparatus 1 used in the vesicle manufacturing method of the present embodiment. As shown in FIG. 1, a vesicle manufacturing apparatus 1 is connected to an outer tube 2a, a double tube 2 composed of an outer tube 2a and an inner tube 2b arranged coaxially in the outer tube 2a. A syringe 3a, a syringe 3b connected to the inner tube 2b, an electrode plate 4 disposed opposite to the double tube 2, and a power supply unit for applying a voltage between the electrode plate 4 and the double tube 2 5, a stirrer 6 disposed on the electrode plate 4, and a container 7 disposed on the stirrer 6.

  The syringe 3a connected to the outer tube 2a is filled with a phospholipid solution 10. The phospholipid solution 10 is a solution in which phospholipid is dissolved in an organic compound solvent. As the organic compound solvent, an organic compound having a boiling point at atmospheric pressure of 110 ° C. or more and having a polarity and being soluble in water is used. Specifically, it is preferable to use polyethylene glycol (PEG), particularly low molecular weight PEG having a molecular weight of 100 to 600, more preferably 200 to 400. This is because it is difficult to dissolve the phospholipid if the molecular weight is too large. Specific examples of the phospholipid include, but are not limited to, lecithin and dicetyl phosphate (DCP). The phospholipid may or may not have a charge.

  A syringe 3b connected to the inner tube 2b is filled with a first liquid 11 made of an aqueous solution or water. The aqueous solution used for the first liquid 11 is preferably an electrolyte aqueous solution or a polar solute aqueous solution, but is not limited to these aqueous solutions. As the electrolyte, a polymer electrolyte is particularly preferable. The polyelectrolyte is a polymer that can be dissociated into a polyion (polycation or polyanion) having a positive or negative charge and a number of low molecular ions having a charge opposite to that of the polyion. The polyelectrolyte used in the first liquid 11 may be a polyanionic polymer (polyanion having a polycation) such as polyallylamine hydrochloride (PAH) or chitosan. A polymer electrolyte). Further, the electrolyte used for the first liquid 11 may be one kind or plural kinds. However, when a plurality of types of polymer electrolytes are used in combination, it is preferable to use polyionic polymers having the same sign.

  In addition, a predetermined substance (hereinafter referred to as a core substance) to be included in the vesicle is added to the first liquid 11. Examples of the core substance include cells, drugs, enzymes, functional foods, fragrances, insecticides, attractants, biocatalysts, proteins, and nucleic acids. The core substance does not necessarily have to be added.

  The container holds the second liquid 12 made of an aqueous solution or water. For example, when polyallylamine hydrochloride is used as the first liquid 11, the second liquid 12 is preferably a citric acid aqueous solution, a phosphoric acid aqueous solution, or the like. The aqueous citric acid solution and the aqueous phosphoric acid solution serve as a buffer solution for keeping the pH of the second liquid 12 containing vesicles constant and making it less susceptible to external and internal factors.

  The inner tube 2b and the outer tube 2a may be a conductive material such as metal or a non-conductor such as glass. The inner diameter of the inner tube 2b is 10 to 1000 μm. The inner diameter of the outer tube 2a is, for example, 1.5 to 10 times the inner diameter of the inner tube 2b. The tip of the inner tube 2b is arranged at a position aligned with the tip of the outer tube 2a.

  The pushers of the syringes 3b and 3b are pressed by two drive units (not shown). The pushing speed of the pusher by the two driving units can be controlled. Therefore, the flow rate of the first liquid 11 pushed out from the inner tube 2b and the flow rate of the phospholipid solution 10 pushed out from the outer tube 2a (specifically, pushed out between the outer tube 2a and the inner tube 2b) are respectively Control is possible.

  Further, the outer tube 2a is connected to the power supply unit 5 so that a high voltage is applied. When the first liquid 11 is a polycationic polymer electrolyte aqueous solution, the positive electrode of the power supply unit 5 is connected to the outer tube 2a, and when the first liquid 11 is a polyanionic polymer electrolyte aqueous solution, the power supply unit 5 is connected to the outer tube 2a. FIG. 1 shows the case where the positive electrode of the power supply unit 5 is connected to the outer tube 2a.

  The electrode on the opposite side to the electrode connected to the outer tube 2a of the power supply unit 5 is grounded and connected to the electrode plate 4 made of a conductive material such as metal. As the power supply unit 5, a DC power supply is used, and the voltage applied to the outer tube 2a (potential difference between the double tube 2 and the electrode plate 4) is, for example, 5 to 30 kV.

  The stirrer 6 is for stirring the second liquid 12 in the container 7. The stirrer 6 is of a magnetic type, for example, and is configured to stir the second liquid 12 by rotating a stirrer disposed in the container 7 by a magnetic force. The material of the container 7 may be conductive or an insulating material.

  Next, a method for producing a vesicle using the vesicle production apparatus 1 will be described with an example in which a polycationic polymer electrolyte aqueous solution is used as the first liquid 11.

  The pushers of the syringes 3a and 3b are pressed to push out the phospholipid solution 10 from the outer tube 2a at a constant speed, and at the same time push out the first liquid 11 from the inner tube 2b. At this time, a voltage is applied between the outer tube 2 a and the electrode plate 4 to form an electric field between the double tube 2 and the electrode plate 4. Due to the influence of this electric field, as shown in FIG. 2, the organic compound solvent in the phospholipid solution 10 pushed out from the outer tube 2a is polarized, so that the surface of the double tube 2 is charged with positive electricity. A conical meniscus is formed.

  At this time, due to the influence of the electric field, as shown in FIG. 2, polycations collect in the first liquid 11 pushed out from the inner tube 2b. Thereby, the polarization of the organic compound solvent of the phospholipid solution 10 pushed out from the outer tube 2a becomes larger. Therefore, the electrostatic force on the surface of the meniscus becomes larger. Note that when a polar solute aqueous solution is used as the first liquid 11, polarization occurs in the portion pushed out from the inner tube 2 b instead of collecting ions.

  When the electrostatic repulsion force exceeds the surface tension, the tip of the meniscus is separated into double-structured microdroplets in which the first liquid 11 (including the core material) is covered with the phospholipid solution 10. The microdroplets are uniform in size due to the action of electrostatic repulsion and surface tension. Since the surface of the separated microdroplet is charged by the polarization of the organic compound solvent, an electrostatic repulsive force is generated between the droplets. Therefore, the fine droplets are ejected in the form of a spray and do not coalesce during movement.

  The ejected micro droplets are attracted to the electrode plate 4 by electrostatic force and dropped onto the second liquid 12. When the microdroplet is brought into contact with the second liquid 12, the organic compound solvent present on the surface side of the microdroplet is dissolved in the second liquid 12. Thereby, since the phospholipid comes into contact with water, a phospholipid bilayer membrane is formed, and a vesicle containing the first liquid 11 (including the core substance) is manufactured.

  When a buffer solution such as phosphoric acid is used as the second liquid 12, the pH of the second liquid 12 containing vesicles is kept constant, and is less susceptible to external and internal factors.

The average particle diameter of the vesicles produced by the above steps is, for example, about 1 to 1000 μm, and the thickness of the phospholipid bilayer membrane is, for example, 5 to 1000 nm.
The particle size and film thickness of the vesicle are adjusted by adjusting the inner diameter of the tubes 2a and 2b, the flow rate of the liquid extruded from the tubes 2a and 2b, the voltage, the type and concentration of the phospholipid, the type and concentration of the first liquid 11, and the like. Can be adjusted.

According to the vesicle manufacturing method described above, a vesicle is manufactured by a simple operation of pushing out the phospholipid solution 10 and the first liquid 11 from the outer tube 2a and the inner tube 2b, respectively, while applying a voltage to the outer tube 2a. be able to. Furthermore, since the vesicle can be continuously produced by continuously extruding from the outer tube 2a and the inner tube 2b at a constant flow rate, the productivity is excellent.
In addition, since the first liquid 11 pushed out from the inner pipe 2b of the double pipe 2 is encapsulated in microdroplets, the first liquid 11 is hardly wasted and sealed in the vesicle at a high sealing rate. Can do.
In addition, if an organic compound solvent that volatilizes at room temperature is used as a solvent for dissolving phospholipid, when the vesicle is continuously produced, the solvent is volatilized and solidified on the tip of the double tube 2. However, it becomes difficult to eject a double droplet having a double structure. On the other hand, in the present embodiment, since a non-volatile organic compound solvent is used at room temperature, no deposit is attached to the tip of the double tube 2, and the vesicle can be manufactured continuously and stably. .

  In addition, when PEG is used as the solvent of the phospholipid solution 10, since PEG is almost harmless, it is possible to produce a vesicle having high safety for a living body. Moreover, phospholipid can be dissolved more reliably by using low molecular weight PEG having a molecular weight of 100 to 600.

  In addition, when an aqueous electrolyte solution or an aqueous solution of a solute having polarity is used as the first liquid 11, charges are collected in the first liquid 11 pushed out from the inner tube 2 b due to the influence of the electric field. The polarization of the organic compound solvent in the phospholipid solution 10 pushed out from is increased. Therefore, since the electrostatic force on the surface of the meniscus becomes larger, the vesicle can be manufactured more reliably and a vesicle having a smaller diameter can be manufactured.

  When a polymer electrolyte aqueous solution is used as the first liquid 11, the first liquid 11 is dissociated into a polyion having a positive or negative charge and a number of low molecular ions having a charge opposite to that of the polyion. Since it becomes easy to influence, it becomes possible to spray more stably.

  Further, by using the first liquid 11 to which a core substance such as a drug is added, a vesicle enclosing the core substance can be easily manufactured.

  Hereinafter, specific examples of the present invention will be described.

  A microcapsule manufacturing apparatus having the configuration shown in FIG. 1 and the following was prepared. As the inner tube, a luer lock injection needle (needle tip 90 °, round base type) having an outer diameter of 630 μm and an inner diameter of 330 μm (23 G) was used. As the outer tube, a nozzle made by MEC Co., Ltd. having an inner diameter of 1 mm and a needle tip of 90 ° was used. As the syringe, an all plastic disposable syringe (lock type, 5 ml, model H4050-LL) was used. A double tube holder made by MEC Co., Ltd. was used, and an electrode plate and a power source unit used NF-104 of “Lab nanofiber sample preparation device for LAB” manufactured by MEC Co., Ltd. The stirrer used was a mini DC stirrer SW-M01. A stainless steel petri dish was used for the container.

<Example 1>
As a phospholipid solution, a solution obtained by dissolving 1 wt% of soybean-derived L-α-lecithin in polyethylene glycol (PEG) having a molecular weight of 200 was used. In addition, 1 μg / ml of a fluorescent labeling substance, L-α-Phosphatidylethanolamine-N- (Lissamine rhodamine Bsulfonyl) (Ammonium Salt), was dissolved in this phospholipid solution.
As the first liquid, an aqueous solution containing 10 wt% polyallylamine hydrochloride (PAH) and 10 μg / ml calcein as a fluorescent labeling substance was used.
As the second solution, a 5 mM citric acid aqueous solution (pH = 5.5) was used.

The applied voltage to the outer tube is 15 kV, the flow rate of the phospholipid solution pushed out from the outer tube is 0.5 ml / h, and the flow rate of the first solution (polymer electrolyte aqueous solution) pushed out from the inner tube is 1.0 ml / h. Then, the distance from the tip of the double tube to the liquid level in the container was 5.0 cm, and a vesicle was produced at room temperature according to the procedure described in the above embodiment. Table 1 shows part of the vesicle manufacturing conditions of Example 1 described above.
The types of phospholipid and organic compound solvent in the phospholipid solution, the distance from the tip of the double tube to the liquid level in the container, and the temperature conditions were all the same in Examples 2 to 11 described later.

<Example 2>
As shown in Table 1, vesicles were prepared under the same conditions as in Example 1 except that the concentration of citric acid in the second solution was 100 mM. The pH of the second liquid (citric acid aqueous solution) is 5.5 as in Example 1.

<Examples 3 to 5>
As shown in Table 1, vesicles were prepared under the same conditions as in Example 1 except that the concentration of PAH in the first solution was 1 wt% and the concentration of citric acid in the second solution was changed. In all of Examples 3 to 5, the pH of the second solution (citric acid aqueous solution) is 5.5 as in Example 1.

<Examples 6 and 7>
As shown in Table 1, a vesicle was prepared under the same conditions as in Example 1 except that an aqueous phosphoric acid solution was used as the second liquid and the concentration of phosphoric acid was changed. In both Examples 6 and 7, the pH of the second liquid (phosphoric acid aqueous solution) is 7.4.

<Example 8>
As shown in Table 1, vesicles were prepared under the same conditions as in Example 7 except that the applied voltage was 20 kV.

<Examples 9 and 10>
As shown in Table 1, vesicles were produced under the same conditions as in Example 7 except that the flow rate of the liquid extruded from the double pipe was different from that in Example 7.

<Example 11>
As shown in Table 1, the concentration of phospholipid (soybean-derived L-α-lecithin) in the phospholipid solution was 10 wt%, and an aqueous solution of 5 wt% chitosan containing 200 mM acetic acid was used as the first liquid. Further, the flow rate of the phosphoric acid solution pushed out from the outer tube is 2.0 ml / h, the flow rate of the first liquid pushed out from the inner tube is 0.2 ml / h, and other conditions are the same as in Example 7, and the vesicle is used. Produced.

  Confocal micrographs of the produced vesicles of Examples 1 to 11 are shown in FIGS. FIGS. 3 (a) to 12 (a) and 13 are photographs showing a state in which rhodamine B is emitted, and FIGS. 3 (b) to 12 (b) are photographs in a state where calcein is emitted. is there.

  In Example 9 (FIG. 10), although the granular thing which is not bag shape was confirmed, the vesicle which includes the 1st liquid has been confirmed in the other Example.

  The vesicle of Example 1 had a particle size of 1 to 8 μm, and the phospholipid bilayer membrane thickness was about 1/10 of the particle size. Moreover, the vesicle of Example 1 was taken out from the container, and this was filtered with a syringe filter (manufactured by Sartorius Co., Ltd.) having a pore diameter of 0.45 μm, and the fluorescence intensity of the filtrate was measured to measure the encapsulation rate. The encapsulation rate was 82%.

  From the results of Examples 1 to 4 (FIGS. 3 to 6), it was confirmed that vesicles could be produced in a wide range of PAH concentrations of 1 to 10 wt%.

  From the comparison of the result of Example 7 (FIG. 9) and the result of Example 8 (FIG. 10), it was confirmed that the higher the applied voltage, the smaller the particle size of the vesicle.

  From the results of Examples 7, 9, and 10 (FIGS. 9, 11, and 12), by changing the ratio of the extrusion flow rate of the inner tube and the outer tube, the particle size of the vesicle and the film thickness of the phospholipid bilayer membrane are changed. I confirmed that I was able to.

  Further, from the results of Examples 7 and 11 (FIGS. 9 and 13), it was confirmed that vesicles could be produced in a wide range of phospholipid concentrations of 1 to 10 wt%.

DESCRIPTION OF SYMBOLS 1 Vesicle manufacturing apparatus 2 Double pipe 2a Outer pipe 2b Inner pipe 3a Syringe 3b Syringe 4 Electrode board 5 Power supply part 6 Stirrer 7 Container 10 Phospholipid solution 11 1st liquid 12 2nd liquid

Claims (7)

Use a double pipe having an outer pipe and an inner pipe arranged coaxially in the outer pipe,
A phospholipid solution in which a phospholipid is dissolved in an organic compound solvent having a boiling point of 110 ° C. or higher at atmospheric pressure and having a polarity and being soluble in water is extruded from the outer tube and at the same time containing water. Extruding one liquid from the inner tube;
The phosphor is pushed out of the outer tube by disposing an electrode opposite to the double tube and applying a voltage to the outer tube to form an electric field between the double tube and the electrode. A step of polarizing a lipid solution to eject fine droplets composed of the first liquid and the phospholipid solution;
The ejected microdroplet is brought into contact with a second liquid containing water, and the organic compound solvent of the microdroplet is dissolved in the second liquid, thereby bringing the phospholipid into contact with the second liquid. Forming a phospholipid bilayer, and producing a microscale vesicle encapsulating the first liquid;
A vesicle manufacturing method characterized by comprising:   The vesicle manufacturing method according to claim 1, wherein the organic compound solvent is polyethylene glycol.   The method for producing a vesicle according to claim 2, wherein the molecular weight of polyethylene glycol constituting the organic compound solvent is 100 to 600.   The vesicle manufacturing method according to any one of claims 1 to 3, wherein the first liquid is an aqueous electrolyte solution or an aqueous solution of a solute having polarity.   The vesicle manufacturing method according to claim 4, wherein the aqueous electrolyte solution constituting the first liquid is a polymer electrolyte aqueous solution.   6. A vesicle containing the first liquid and the predetermined substance is manufactured by adding a predetermined substance in advance to the first liquid pushed out from the inner tube. A vesicle manufacturing method according to any one of the above.   The predetermined substance includes at least one of a cell, a drug, an enzyme, a functional food, a fragrance, an insecticide, an attractant, a biocatalyst, a protein, and a nucleic acid. Vesicle manufacturing method.