JPWO2007032225A1 - Liposome, liposome production method, and reaction control method in minute reaction space - Google Patents

Abstract

Provided are a micrometer-sized liposome whose inner membrane is composed of only one or more selected from the group of PS and PE, a method for producing the liposome, and a reaction control method. An inner membrane phospholipid is a liposome having a particle size of 5 μm or more formed of a lipid bilayer membrane, comprising only one or more selected from the group of phosphatidylserine and derivatives thereof, and phosphatidylethanolamine and derivatives thereof. Thus, a cytoskeletal polymer is encapsulated in the inner membrane phospholipid. [Selection] Figure 1

Description

  The present invention relates to a liposome, a method for producing a liposome, and a reaction control method in a minute reaction space.

Liposomes are formed by lipid bilayers such as phospholipids and have a capsule structure in which an aqueous phase is confined. Since the structure of liposomes is similar to biological membranes, various research materials and applications (cosmetics and drug delivery systems (DDS)) containing active ingredients are being studied (for example, Patent Documents 1 and 2). reference).
Here, liposomes are usually produced by suspending phospholipids in an aqueous solution to form multilamellar liposomes (so-called liposome stock solutions), followed by sonication.

On the other hand, a liposome production method has been proposed in which the inner membrane of a liposome is made of a W / O emulsion and lipid is added to the outer membrane of this emulsion (see, for example, Non-Patent Document 1). This method is performed as follows.
First, a small amount of water and lipid (which becomes the inner membrane of a liposome) are mixed in oil to prepare a W / O emulsion. Next, an oily liquid in which an outer membrane lipid (which becomes an outer membrane of a liposome) is dissolved is added to the aqueous phase to form a molecular membrane in which outer membrane lipids are arranged at the separation interface of oil and water. Then, the W / O emulsion is added to the oil phase side at the separation interface, centrifuged or stirred, and the W / O emulsion is transferred to the water phase side at the separation interface, and the outer membrane lipid is placed outside the W / O emulsion. Is added to produce liposomes.

By the way, when studying the function of a biological substance in a cell, it is desired to construct a microlab (μm size reaction field) having almost the same spatial scale and structure as a cell. Here, it is inconvenient if the particle size of the microlab is too small to handle the obtained microlab to various solvents, etc., or to observe the biological material in the microlab with a laser tweezer. In consideration of the above, it is necessary that the particle size of the microlab is about 5 μm or more.
As such a microlab, a liposome whose structure is similar to that of a biological membrane can be used. For example, there is a microlab made of acrylic resin or glass. In addition, there is a technique in which a μm sized W / O emulsion is prepared with a surfactant such as sodium dodecyl sulfate (SDS), and a reaction substrate is enclosed in the emulsion to form a reaction field.

  On the other hand, techniques for encapsulating actin filaments (cytoskeleton) in liposomes have been reported (for example, see Non-Patent Documents 2 and 3).

Japanese Patent Laid-Open No. 2003-2824 JP 2005-162712 A Langmuir 19 (2003) 2870-2879 Proc Natl Acad Sci USA, (1992) December 1; 89 (23), 11547.1551 Eur Phyd J E soft Matter (2003) Apr; 10 (4) .319-30

  However, in the case of a conventional method for producing a liposome having a particle size of 5 μm or more, the inner membrane of the liposome is phosphatidylserine (hereinafter referred to as “PS”) or phosphatidylethanolamine (hereinafter referred to as “PE”). It is difficult to produce liposomes consisting only of species. This is thought to be due to the large negative spontaneous curvature of PS and PE. Therefore, in the past, liposomes were produced by mixing phosphatidylcholine (hereinafter referred to as “PC”), which is a lipid having a small negative spontaneous curvature, and the above-mentioned PS or PE. A liposome that is a single layer of PS or PE cannot be obtained.

  On the other hand, in the case of the technique described in Non-Patent Document 1, it is difficult to produce liposomes having a particle size of 5 μm or more. This is presumably because the liposome has a very unstable structure, and when it becomes larger than 5 μm, the liposome is broken at the oil-water interface by centrifugation or the like.

Moreover, those using acrylic resin or glass as a microlab have a problem that these surfaces deactivate or denature biological substances. Further, when a W / O emulsion is used as a microlab, the surfactant used in the emulsion deactivates or denatures the biological material.
As a microlab, a micrometer-sized liposome composed of the same lipid bilayer membrane as the cell membrane can be used, and the reaction proceeds inside the liposome without inactivating the biological substance. However, since liposomes have an unstable structure, they are easily deformed and broken under the influence of an external field such as osmotic pressure, making it difficult to control the reaction. In addition, lipids that can be produced as the inner membrane of the liposome are limited, and it is difficult to control the internal environment of the microlab by changing the type of lipid.

In addition, the techniques described in Non-Patent Documents 2 and 3 for encapsulating actin in liposomes encapsulate actin monomers in the liposome in advance, and the temperature is increased (Non-patent Document 2) or polymerizing agent (Non-Patent Document 3) ) To form actin filaments. This is because it is difficult to encapsulate actin filaments, which are macromolecules, in the liposome, but the lipid used as the liposome is limited in the same manner as described above, and PS and PE cannot be used. The main components of the normal inner membrane are PS and PE, and function in an environment where the cytoskeleton (here, actin) is surrounded by the inner membrane. Therefore, when the cytoskeleton is encapsulated in the microlab, it is expected that the compatibility with the cytoskeleton is better when PS or PE is used as the lipid.
Furthermore, in the case of the techniques described in Non-Patent Documents 2 and 3, there is a problem that the concentration of the salt used for the polymerization of the actin filament and the buffer cannot be freely selected. For example, in Non-Patent Documents 2 and 3, G-buffe (actin buffer is often used as an actin buffer: 2 mM Tris HCl, pH 8.2, 0.5 mM CaCl2, 0.2 mM ATP, 0.2 Although it is described that mM NaN3, 0.5 mM dithiothreitol) is used, it is unclear whether liposomes are formed when the buffer concentration is outside this range. This is because liposomes usually tend to be difficult to produce when the salt concentration is high, and therefore the usable salt concentration is limited.
When liposomes are used for DDS in the future, it is advantageous that there is no restriction on the solute concentration as a liposome preparation condition because the drug is encapsulated in the liposome at a high concentration.

  Accordingly, an object of the present invention is to provide a micrometer-sized liposome whose inner membrane is composed of only one or more selected from the group of PS and PE (including these derivatives), and a method for producing the same. Another object of the present invention is to provide a reaction control method in a micro reaction space of micrometer size.

  That is, the liposome of the present invention has an inner membrane phospholipid consisting of only one or more selected from the group of phosphatidylserine and derivatives thereof, and phosphatidylethanolamine and derivatives thereof, and having a particle size of 5 μm or more formed from a lipid bilayer It is a liposome that encapsulates a cytoskeletal polymer in the inner membrane phospholipid.

  The cytoskeleton is preferably an actin filament.

  The liposome production method of the present invention has an inner membrane phospholipid consisting of only one or more selected from the group of phosphatidylserine and derivatives thereof, and phosphatidylethanolamine and derivatives thereof, and has a particle size of 5 μm formed from a lipid bilayer membrane. In the method for producing the above liposome, the inner membrane phospholipid, the cytoskeleton, and the aqueous liquid are mixed in an oily liquid, and the cytoskeleton is bound in a fibrous form in the inner membrane phospholipid. A step of forming a W / O emulsion, a step of adding an oily liquid in which an outer membrane lipid is dissolved to an aqueous phase, and forming a molecular membrane in which the lipids are arranged at an oil-water separation interface; In addition to the oil phase side at the separation interface, centrifugal separation or stirring is performed, and the W / O emulsion is transferred to the water phase side at the separation interface, so that the outside of the W / O emulsion is removed. And a step of adding the outer membrane lipids.

  The cytoskeleton is preferably an actin filament, and the lipid that forms the outer membrane preferably contains phosphatidylcholine or a derivative thereof.

  The method for controlling a reaction in a micro reaction space according to the present invention comprises mixing at least one phospholipid, a reaction substrate, and an aqueous liquid in an oily liquid, and the reaction substrate is enclosed in the phospholipid. A step of forming a W / O emulsion having a particle size of 5 μm or more, and a step of introducing a reactive agent that reacts with the reaction substrate into the W / O emulsion.

  Also, the reaction control method in the minute reaction space according to the present invention comprises mixing at least one phospholipid, a reaction substrate and an aqueous liquid in an oily liquid, and enclosing the reaction substrate in the phospholipid. Forming a first W / O emulsion having a particle size of 5 μm or more, mixing one or more phospholipids, a reactant that reacts with the reaction substrate, and an aqueous liquid in an oily liquid; Fusing the step of forming a second W / O emulsion having a particle size of 5 μm or more in which the reactant is encapsulated in the phospholipid, and the first W / O emulsion and the second W / O emulsion And a step of causing

  In the method for controlling reaction in the microreaction space of the present invention, one or more phospholipids, a reaction substrate, and an aqueous liquid are mixed in an oily liquid, and the reaction substrate is contained in the phospholipid. A step of forming an encapsulated W / O emulsion having a particle size of 5 μm or more, and a step of irradiating the W / O emulsion with wavelength light or electromagnetic waves that react with the reaction substrate.

  According to the present invention, a micrometer-sized liposome whose inner membrane is composed of one or more selected from the group of PS and PE (including these derivatives) can be produced. In addition, according to the present invention, the reaction can be controlled in a micrometer-sized minute reaction space made of phospholipid.

  Hereinafter, embodiments of the present invention will be described.

1. Liposomes of the Present Invention The liposomes of the present invention have an inner membrane phospholipid consisting of only one or more selected from the group of phosphatidylserine and derivatives thereof, and phosphatidylethanolamine and derivatives thereof, and the particle size formed from a lipid bilayer membrane It is a liposome having a size of 5 μm or more, and encapsulating a cytoskeleton polymer in the inner membrane phospholipid.
In this way, since the inner membrane of the liposome is PS and / or PE, it can be expected that compatibility with the encapsulated cytoskeleton is improved.

2. Method for producing liposome according to the present invention
<Mixing process (production of W / O emulsion)>
In the method for producing liposomes of the present invention, first, an inner membrane phospholipid, a cytoskeleton, and an aqueous liquid are mixed in an oily liquid, and the cytoskeleton is bound in a fibrous form in the inner membrane phospholipid. O emulsion (inner membrane phospholipid monomolecular film particles) is formed.
As the inner membrane phospholipid, one comprising one or more selected from the group of PS or a derivative thereof and PE or a derivative thereof is used. Here, as a derivative | guide_body of PS, a dioleyl phosphatidylserine (DOPS) etc. are mentioned. Therefore, in the present invention, the inner membrane phospholipid may be, for example, PS alone or may be composed of PS and PE.

The cytoskeleton is an intracellular fibrous structure that maintains cell morphology and causes movement inside and outside the cell. The cytoskeleton can be observed with an electron microscope, and is classified into three types depending on its thickness. 1) Microfilament (actin filament): a fiber having an outer diameter of about 5 nm, and usually has actin as a structural unit. 2) Intermediate filament: contains various fibers and has an outer fiber diameter of about 10 nm. 3) Microtubule: An outer diameter of the fiber is about 25 nm, and tubulin is usually used as a structural unit.
In the present invention, when the W / O emulsion is prepared, the W / O emulsion is strengthened by confining the cytoskeleton in the aqueous phase inside the inner membrane phospholipid and binding the cytoskeleton in a fibrous form.

In addition, in order to polymerize the cytoskeleton inside the W / O emulsion and bond it in a fibrous form, it is preferable to add a cytoskeleton polymerization agent. The polymerization agent may be mixed when preparing the W / O emulsion. For example, when an actin filament is used as the cytoskeleton, about 2 mmol / L of Mg ²⁺ may be added as a polymerization agent. In this case, actin filaments having a length of about 10 μm are obtained, and actin filaments having these unit lengths are entangled with each other and bind to inner membrane phospholipids to strengthen the W / O emulsion.

Here, when liposomes are used for DDS, since the drug is encapsulated in the liposomes at a high concentration, it is advantageous that there are no restrictions on the solute concentration as the preparation conditions of the liposomes.
In the production method of the present invention, first, a monolayer membrane vesicle is produced, and after increasing the strength with an actin filament, a bilayer membrane vesicle is produced. Can be freely selected (the lower limit of the concentration is 0, that is, it can be produced even with pure water, and can be higher than the conventional one).
The reason for this is considered to be that the influence of surface tension is dominant in the first monolayer vesicle formation stage, and the salt concentration is not particularly limited. On the other hand, when the bilayer membrane vesicles are formed at a time as in the prior art, the influence of the salt concentration is increased in the formation process, and the concentration conditions are considered to be limited.
Furthermore, as will be described later, it has been found that the structure of actin filaments varies depending on the magnesium ion concentration. Therefore, the ability to freely select the salt concentration means that the liposome strength may be controlled by the structure of the actin filament.

In order to form a W / O emulsion having a particle size of 5 μm or more, it is usually preferable to mix the inner membrane phospholipid, the oily liquid, and the aqueous liquid at the following mixing ratio.
1) It mix | blends so that the density | concentration in the oil-based liquid of inner membrane phospholipid may be 1 mmol / L-10 mmol / L. Here, when using 2 or more types as inner membrane phospholipid, let the total density | concentration be the said range.
2) The mixing ratio of the aqueous liquid and the oily liquid is set to be (aqueous liquid / oily liquid) = 1/1000 to 1/10 in volume ratio.

Here, when the concentration of the inner membrane phospholipid in the oily liquid is less than 1 mmol / L, the obtained monomolecular membrane particles may be too small or the monomolecular membrane particles may not be formed. On the other hand, when the concentration exceeds 10 mmol / L, the particle diameter of the monomolecular film particles may be 5 μm or less, or may be lamellar or the like without becoming particulate. In general, the particle size of the W / O emulsion is preferably 40 μm or less.
In addition, when the ratio represented by (aqueous liquid / oil liquid) is less than 1/1000, the ratio of the oil liquid is excessive, and a good W / O emulsion (monolayer film of inner membrane phospholipid) is obtained. It may be difficult to form. On the other hand, if the above ratio exceeds 1/10, the proportion of the aqueous liquid increases too much, and it may be difficult to form a good W / O emulsion.

  The oily liquid is not particularly limited as long as it stably disperses the inner membrane phospholipid. For example, mineral oil (mineral oil) can be used. The aqueous liquid is not particularly limited, and the active ingredient or the like encapsulated in the finally obtained liposome may be contained in the aqueous liquid, but it is easy to control the particle size of the monomolecular film particle (liposome). Water (pure water) is preferred.

<Preparation of liposome (addition of outer membrane lipid)>
Next, a lipid serving as an outer membrane is added to the surface of the monomolecular membrane particle to prepare a liposome having a particle size of 5 μm or more formed from an inner membrane and a lipid bilayer membrane of the outer membrane.
Here, when the particle size of the finally obtained liposome is less than 5 μm, it is difficult to observe the liposome with visible light when the liposome is captured and handled with laser tweezers.
In addition, in order to make the particle size of the finally obtained liposome 5 μm or more, the particle size of the monomolecular membrane is precisely smaller than the particle size of the liposome by the length of the outer membrane lipid. For convenience, it is considered the same as the particle size of the liposome.

  The particle size of the liposome can be measured, for example, with an optical microscope.

Examples of the method for adding a lipid as an outer membrane are described in literature (Langmuir, 19 (2003) p. 2870-2879, and Proc. Natl. Acad. Sci., 101. (2004) p. 17669-17674). Method can be used.
Specifically, 1) An oily liquid in which outer membrane lipids are dissolved is added to the aqueous phase, and a molecular membrane in which the lipids are lined up is formed at the oil-water separation interface. Usually, the oil phase is above the water phase.
2) Next, the W / O emulsion is poured onto the oil phase side at the separation interface of 1). When this is centrifuged or stirred, the W / O emulsion passes through the separation interface and moves to the aqueous phase side. During this passage, outer membrane lipids arranged as molecular membranes are added to the outside of the W / O emulsion.

  As the lipid that forms the outer membrane, for example, PC or a derivative thereof can be used. In order to stabilize the liposome, a membrane stabilizer, a charge-imparting substance, and various auxiliary agents may be added.

3. 3. Reaction control method in minute reaction space according to the present invention 3-1. Embodiment of Introducing Reactant into W / O Emulsion This embodiment includes the following steps.
<Preparation of W / O emulsion>
In the reaction control method of the present invention, first, a W / O emulsion is formed. The method for forming the W / O emulsion is the same as in the above-described method for producing liposomes, and one or more phospholipids, a reaction substrate, and an aqueous liquid are mixed in an oily liquid, and the reaction substrate is a phospholipid. A W / O emulsion having a particle size of 5 μm or more enclosed therein is formed. The phospholipid is not particularly limited, and examples thereof include PS and its derivatives, PE and its derivatives, PC (phosphatidylcholine) and its derivatives, SM (such as sphingomyelin) and its derivatives.
The types that can be used as the oily liquid, the phospholipid, and the aqueous liquid, and the blending ratio thereof are the same as those in the method for producing the liposome.
As the reaction substrate, biological materials for synthesizing various proteins, DNA, and the like, which are biological materials, can be suitably used. The biological material is very sensitive to the surrounding environment, and it is difficult to control the reaction of the biological material in a conventional micrometer-sized microspace. According to the present invention, a biological material can be surrounded by a phospholipid similar to the main component of the cell membrane, and a minute reaction space similar to the intracellular environment can be created inside, so that the reaction can be performed without inactivating the biological material. Can be controlled.

Next, a reactive agent that reacts with the reaction substrate is introduced into the W / O emulsion.
Any reactant that reacts with the reaction substrate may be used. For example, when the reaction substrate is a protein, an enzyme that specifically reacts with the protein becomes a reactive agent.
As a method for introducing the reactant into the W / O emulsion, a method in which a micropipette is inserted into the W / O emulsion by a micromanipulator and the reactant in the pipette is injected.
In this embodiment, the reactant may be enclosed in the W / O emulsion, and the reaction substrate may be introduced into the W / O emulsion from the outside.

3-2. Embodiment of Fusing Multiple W / O Emulsions This embodiment includes the following steps.
<Preparation of first W / O emulsion>
A first W / O emulsion having a particle size of 5 μm or more in which a reaction substrate is encapsulated is formed in an oily liquid. The types that can be used as the oily liquid, the phospholipid, and the aqueous liquid for forming the W / O emulsion, and the blending ratio thereof are the same as those in the above-described liposome production method.
As the reaction substrate, those described in the above embodiment 2-1 can be preferably used.
<Preparation of second W / O emulsion>
A first W / O emulsion having a particle size of 5 μm or more is formed in an oily liquid in which a reactant is encapsulated. Kinds that can be used as oily liquids, phospholipids (PS, PE and their derivatives), and aqueous liquids for forming W / O emulsions, and the blending ratios thereof are the same as in the case of the above-described method for producing liposomes. It is.
As the reactant, those described in the above embodiment 2-1 can be suitably used.

  In this embodiment, instead of introducing the reactant into the W / O emulsion in which the reaction substrate is encapsulated, the first W / O emulsion in which the reaction substrate is encapsulated and the second W / O in which the reactant is encapsulated. It is characterized by fusing with the O emulsion and allowing the reaction to proceed. As a method for fusing the two, there is a method in which the two are previously separated from each other and placed in oil, and both are held and fused by laser tweezers. In this case, for example, the mineral oil can be added to an oil sump having a glass plate as a bottom surface and a plastic side wall rising from the bottom surface, and the W / O emulsion can be poured into the oil. It is preferable to dissolve 0.01 to 1 mM of the lipid used for preparing the emulsion in the mineral oil.

3-3. Embodiment of irradiating W / O emulsion with wavelength light or electromagnetic wave This embodiment is the same as that described above except that the W / O emulsion in which the reaction substrate is encapsulated is irradiated with wavelength light or electromagnetic wave that reacts with the reaction substrate. Since this is the same as the first embodiment, the description thereof is omitted.

<Example>
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
<Examples for the production method of liposome>

DOPS 1 mmol / L as inner membrane phospholipid is dissolved in mineral oil (product number SP 23306-84, manufactured by Nacalai Co., Ltd.), and distilled water is added in an amount of 1/100 by volume with respect to mineral oil. 1 μl of actin solution (1 mg / ml actin and 2 mM magnesium ion) was added and stirred (pipette was repeatedly put in and out of the mixture several times) to prepare a W / O emulsion. Mixing and stirring were performed at room temperature. By leaving it to stand for 2 hours, a W / O emulsion in which actin filaments bound in a fibrous form were encapsulated in DOPS was obtained. It was confirmed with an optical microscope that the particle size of the W / O emulsion was 5 to 40 μm.
Next, 2 mL of mineral oil in which 1 mmol / L of outer membrane lipid (PC) was dissolved was added from above 3 mL of water and allowed to stand for about 90 minutes. Oil-water separated into two phases, and outer membrane lipids were arranged as a monomolecular membrane at the oil-water separation interface. The upper side of the separation interface was the oil phase and the lower side was the water phase.
1 mL of the above W / O emulsion was poured into the oil phase at the oil / water separation interface, and the whole liquid was centrifuged (120 g of liquid for 5 to 10 minutes). When the emulsion passed through the oil-water interface, outer membrane lipid was added and liposomes were obtained.

  Liposomes with a particle size of 5 to 40 μm were obtained in exactly the same manner as in Example 1 except that DOPE 10 mmol / L was used instead of DOPS as the inner membrane phospholipid.

<Comparative Example 1>
A W / O emulsion was prepared in the same manner as in Example 1 except that the actin solution was not added, and outer membrane lipid (PC) was added thereto. When the W / O emulsion was poured into the oil phase at the oil-water separation interface and the liquid was centrifuged, the emulsion was broken and liposomes having a particle size of 5 μm or more could not be obtained.

As is clear from the above, in the case of each Example, liposomes having a particle size of 5 to 40 μm in which the inner membrane phospholipid was composed only of DOPS or DOPE and the outer membrane was PC were obtained.
FIG. 1 shows the suspension of Example 1. It can be seen that an emulsion in which the inner membrane phospholipid was dispersed as monomolecular membrane particles 2 having a particle size of about 10 μm was formed.

  On the other hand, in the case of Comparative Example 1 in which no actin solution was added, liposomes having a particle size of 5 μm or more were not obtained. This is presumably because the cytoskeleton was not bound to the inside of the W / O emulsion (aqueous phase) in a fibrous form, and the strength of the emulsion was not improved, so that the emulsion was destroyed by centrifugation.

<Structural change of polymer in W / O emulsion of DOPS>
In order to show that the inside of the above-mentioned DOPS monomolecular film particles can be used as a micrometer-sized reaction field, a W / O emulsion (using DOPS) containing actin filaments was prepared in the same manner as in Example 1, and actin was used. The structural change of was observed. Actin filaments are known to have an aggregated structure in the presence of a polyvalent cation such as magnesium ion.
In addition, the actin filament was fluorescently red using a fluorescent substance, and the structure of the actin filament was observed using a confocal microscope.

FIG. 2 shows the structure of actin filaments in monolayer particles. In FIG. 2, the Mg ²⁺ concentration is 4 mmol / L (cation conversion), and the actin filaments 8 are uniformly distributed in the monomolecular film particles 4. Since actin filaments are negatively charged polymers and the DOPS film is also negatively charged, it is thought that both do not aggregate due to electrostatic repulsion.
FIG. 3 shows the case where Mg ²⁺ is set at a concentration of 12 mmol / L (cation conversion). It can be seen that the actin filaments 8 are aggregated and transferred to the membrane.
FIG. 4 shows the case where Mg ²⁺ is 40 mmol / L (cation conversion). It can be seen that the actin filament 8 undergoes bundle transfer and adhesion to the membrane is reduced. When bundle transition occurs, the attachment of a thick and hard bundle of actin filaments to a DOPS film having a curvature is accompanied by energy loss, so that the adhesion to the film is considered to have decreased.
In addition, when DOPE was used instead of DOPS as the monomolecular film particle, the structural change of the actin filament could be observed similarly.
<Example of reaction control method in minute reaction space>

Calcein AM (bis [N, N-bis (carboxymethyl) aminomethyl] fluorescene) was used as a reaction substrate. In addition, an esterase enzyme that hydrolyzes calcein AM to generate fluorescent calcein was used as a reactant.
A W / O emulsion was prepared in exactly the same manner as in Example 1 except that calcein AM was added instead of adding the actin solution to the mineral oil. The amount of calcein AM added was adjusted so that the calcein AM in the W / O emulsion was 50 μmol / L.
The mineral oil was added to an oil sump with a glass plate as the bottom and a plastic side wall raised from the bottom, and the W / O emulsion was charged into the oil. Next, using a micromanipulator (Narishige Microinjector IM-300), a micropipette (tip diameter of 1 to 2 μm) was inserted into the W / O emulsion, and the esterase enzyme (2.5 mg / ml) in the pipette. ) Was injected.
FIG. 5 shows a mode in which the pipette 12 is injected into the W / O emulsion 10. In this figure, the enzyme in the pipette 12 reacts with calcein AM10a in the W / O emulsion.

  Moreover, the state of the W / O emulsion after inject | pouring esterase enzyme is shown in FIG. 6A shows a state immediately after the injection, and FIG. 6B shows a state 24 minutes after the injection. The emulsion 10 in FIG. 6B was shining, and from this, it was confirmed with a fluorescence microscope that a chemical reaction to calcein emitting fluorescence progressed. As described above, for the first time in the present invention, the reaction could be controlled without denaturing the protein in a micrometer-sized reaction field.

The same reaction substrate and reaction agent as in Example 4 were used.
First, in the same manner as in Example 4, a first W / O emulsion in which calcein AM was encapsulated was prepared. In addition, a second W / O emulsion in which the esterase enzyme was encapsulated was prepared in exactly the same manner as in Example 4 except that esterase enzyme was added instead of calcein AM.

The first and second W / O emulsions were placed separately in oil sumps made of the mineral oil. Next, using a laser tweezers (Nd: YAG laser, Spectron SL902T, wavelength is 1064 nm), the first and second W / O emulsions were respectively held and fused together.
FIG. 7 shows a state where the first W / O emulsion 20 and the second W / O emulsion 30 are placed separately on the oil sump 40.

  A state in which the first and second W / O emulsions are fused is shown in FIG. In this figure, FIG. 8 (a) shows the first W / O emulsion 20, the second W / O emulsion 30 and the laser-tweezers (laser spot) 50 before fusion. FIG. 8B shows a state during fusion, and FIG. 8C shows a state after fusion. Also in this example, it was confirmed by a fluorescence microscope that a chemical reaction to calcein emitting fluorescence progresses. Therefore, for the first time in the present invention, the reaction could be controlled without denaturing the protein in a micrometer-sized reaction field.

A biological material for synthesizing protein (GFP) was used as a reaction substrate. This biological material is composed of a large number of biological materials necessary for gene expression, such as all 20 types of amino acids and ribosomes. In the experiment, a kit of “E.coli S30 Extract System for circular DNA” manufactured by Promega was used.
As a reaction agent, a gene (Promega's E. coli S30 Extract System for circular DNA) that synthesizes protein (GFP) was used.
A W / O emulsion was prepared in exactly the same manner as in Example 1 except that the above gene and amino acid were added instead of adding the actin solution to the mineral oil. Next, the gene was expressed in the W / O emulsion to synthesize protein (GFP). It was confirmed with a fluorescence microscope that a fluorescent protein (GFP) was synthesized. FIG. 9 shows a state in which the protein (GFP) emits fluorescence in the W / O emulsion 10. FIG. 9 is a fluorescence microscopic image after leaving the emulsion at 37 degrees for 1 hour.

  As described above, since the micrometer-sized reaction field (W / O emulsion) used in Examples 4 to 6 is similar to the intracellular environment, the biological material typified by protein is not denatured and reacted. It is also excellent in that it can be controlled. Furthermore, since the capacity of this reaction field is about one trillionth of that of a normal test tube, biochemical experiments in which trial and error are forced by a combination of many experiments, various blood tests, pharmaceutical production or In the efficacy test, etc., the amount of reagents and blood required is small, and it is very useful for low cost and examinations that do not burden the patient.

2 is a view showing a micrograph of a suspension of Example 1. FIG. It is a figure which shows the microscope picture of the structure of the actin filament in a monomolecular film particle. It is another figure which shows the microscope picture of the structure of the actin filament in a monomolecular film particle. It is another figure which shows the microscope picture of the structure of the actin filament in a monomolecular film particle. It is a figure which shows the aspect which inject | pours a pipette into W / O emulsion. It is a figure which shows the state of the W / O emulsion after inject | pouring esterase enzyme. It is a figure which shows the state which spaced apart and mounted the 1st W / O emulsion and the 2nd W / O emulsion 30 on the glass plate. It is a figure which shows the state which the 1st and 2nd W / O emulsion united. It is a figure which shows the state which protein (GFP) fluoresces within a W / O emulsion.

Explanation of symbols

2, 4, 10, 20, 30 W / O emulsion (monomolecular film particles)
8 Actin filament

Claims (9)

The inner membrane phospholipid is composed of only one or more selected from the group of phosphatidylserine and derivatives thereof, and phosphatidylethanolamine and derivatives thereof, and is a liposome having a particle size of 5 μm or more formed from a lipid bilayer membrane,
Liposomes encapsulating a cytoskeleton polymer in the inner membrane phospholipid.   The liposome according to claim 1, wherein the cytoskeleton is an actin filament. This is a method for producing a liposome having a particle size of 5 μm or more, wherein the inner membrane phospholipid comprises only one or more selected from the group of phosphatidylserine and its derivatives, and phosphatidylethanolamine and its derivatives, and is formed from a lipid bilayer membrane. And
Mixing the inner membrane phospholipid, the cytoskeleton, and an aqueous liquid in an oily liquid to form a W / O emulsion in which the cytoskeleton is bound fibrously in the inner membrane phospholipid;
Adding an oily liquid in which an outer membrane lipid is dissolved to the aqueous phase, and forming a molecular membrane in which the lipids are arranged at the separation interface of oil and water;
The W / O emulsion is added to the oil phase side at the separation interface, centrifuged or stirred, and the W / O emulsion is transferred to the water phase side at the separation interface, so that the outside is outside the W / O emulsion. A method for producing a liposome, comprising a step of adding a membrane lipid.   The method for producing a liposome according to claim 3, wherein the cytoskeleton is an actin filament.   The method for producing a liposome according to claim 3 or 4, wherein the lipid serving as the outer membrane contains phosphatidylcholine or a derivative thereof. Mixing one or more phospholipids, a reaction substrate and an aqueous liquid in an oily liquid to form a W / O emulsion having a particle size of 5 μm or more in which the reaction substrate is enclosed in the phospholipid; ,
A method for controlling a reaction in a minute reaction space, which comprises a step of introducing a reactive agent that reacts with the reaction substrate into the W / O emulsion. One or more phospholipids, a reaction agent that reacts with a reaction substrate, and an aqueous liquid are mixed in an oily liquid, and the W / O emulsion having a particle size of 5 μm or more is encapsulated in the phospholipid. Forming a step;
A method for controlling a reaction in a minute reaction space, comprising the step of introducing the reaction substrate into the W / O emulsion. One or more phospholipids, a reaction substrate and an aqueous liquid are mixed in an oily liquid to form a first W / O emulsion having a particle size of 5 μm or more in which the reaction substrate is enclosed in the phospholipid. And a process of
In an oily liquid, one or more phospholipids, a reaction agent that reacts with the reaction substrate, and an aqueous liquid are mixed, and a second particle having a particle size of 5 μm or more enclosed in the phospholipid. Forming a W / O emulsion;
A reaction control method in a minute reaction space, comprising the step of fusing the first W / O emulsion and the second W / O emulsion. A step of mixing one or more phospholipids, a reaction substrate and an aqueous liquid in an oily liquid to form a W / O emulsion having a particle size of 5 μm or more in which the reaction substrate is enclosed in the phospholipid. When,
A method for controlling reaction in a minute reaction space, which comprises irradiating the W / O emulsion with light of a wavelength or electromagnetic wave that reacts with the reaction substrate.