METHOD FOR PRODUCING LIPOSOME BY cDICE METHOD AND DEVICE THEREFOR

Abstract

To provide a method for producing liposome by a cDICE method capable of producing liposome more simpler than a well-known method and a device therefor.SOLUTION: A method for producing liposomes includes a step of forming a liposome external liquid layer in the outer region of a rotatable chamber, a step of forming a lipid liquid layer in the inner region of the chamber that contacts the liposome external liquid layer, and a step of supplying liposome internal droplets to the lipid liquid layer while rotating the chamber, allowing the liposome internal droplets to pass through the interface between the liposome external liquid layer and the lipid liquid layer due to centrifugal force, thereby generating liposomes enclosed by a lipid bilayer within the liposome external liquid layer. It also includes a step of supplying the liposome internal liquid near the rotation axis of the chamber, and using centrifugal force to cause the liposome internal liquid to pass through pores and supply liposome internal droplets to the lipid liquid layer, and a step of further moving the supplied liposome internal droplets outward through the interface due to centrifugal force.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for producing liposomes by the cDICE method and an apparatus therefor. [Background technology]

[0002] Cell-sized liposomes (also known as "giant liposomes") are expected to be used in the production of organic compounds that cannot be synthesized organically, cell therapy, sensors, and other applications, and basic research into them as artificial cell materials of the future is being actively conducted. The main method used to manufacture liposomes containing biomolecules is the interface passing method, in which lipid-dispersed water droplets in oil are passed through an oil-water interface to be covered with a lipid bilayer membrane. The interface passing method can be implemented using a centrifuge or a rotating device such as a spin coater, called cDICE ( c continuous D roplet I interface C Rossing E One method is called uncapsulation (continuous droplet interface cross-encapsulation).

[0003] To form liposomes using the interface passing method using a centrifuge, it is necessary to consider the conditions for each molecule to be encapsulated in the liposomes. However, when setting the centrifugal force, i.e., the rotation speed, it is difficult to eliminate the velocity gradient caused by acceleration from a stationary state, making it difficult to consider the conditions precisely. On the other hand, the cDICE method using a spin coater has the advantage of overcoming this problem. However, the conventional cDICE method requires the operation of a glass capillary and a microfluidic pump to form water-in-oil droplets, which is cumbersome. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2012-533421 [Non-patent literature]

[0005] [Non-Patent Document 1] Abkarian, Manouk, Etienne Loiseau, and Gladys Massiera. "Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design." Soft Matter 7.10 (2011): 4610-4614. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present invention is to provide a novel method for producing liposomes by the cDICE method, which can produce liposomes more easily than known methods, and an apparatus therefor. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that giant liposomes can be easily produced by supplying intraliposomal droplets from near the center of rotation of a chamber through a through hole to the lipid liquid layer by centrifugal force in the cDICE method, and have completed the present invention. Furthermore, they have found that by passing through a through hole, the diameter of the produced liposomes is uniform to be equal to or smaller than the diameter of the through hole.

[0008] That is, the present invention provides the following. (1) forming an outer liposome liquid layer in an outer region within a rotatable chamber; forming a lipid liquid layer in contact with the liposome external liquid layer in an inner region of the chamber; A method for producing liposomes by a cDICE method, comprising the steps of: supplying intraliposomal droplets to the lipid liquid layer while rotating the chamber; and passing the intraliposomal droplets through an interface between the liposomal external liquid layer and the lipid liquid layer by centrifugal force, thereby generating liposomes surrounded by a lipid bilayer membrane in the liposomal external liquid layer, A method for producing liposomes, comprising the steps of supplying a liposome internal liquid near a rotation axis of the chamber, causing the liposome internal liquid to pass through one or more holes by centrifugal force to supply the liposome internal liquid droplets to the lipid liquid layer, and moving the supplied liposome internal liquid droplets further outward by centrifugal force to pass through the interface. (2) The method according to (1), further comprising: placing a cylindrical intraliposomal droplet generating vessel having one or more through holes on its side surface such that the center of the circle of the cross section of the cylinder coincides with the rotation axis; feeding the intraliposomal liquid into the intraliposomal droplet generating vessel while the chamber is rotating; and supplying the intraliposomal liquid to the lipid liquid layer by passing the intraliposomal liquid droplets through the through holes on the side surface of the intraliposomal droplet generating vessel. (3) The method according to (2), wherein the container has a penetrating opening on its side, and the opening is covered by the membrane having the perforated hole. (4) The method according to (3), wherein the membrane is a track-etched membrane having a plurality of the holes. (5) The method according to (4), wherein the through hole has a diameter of 1 μm to 20 μm. (6) A liposome production apparatus for the cDICE method, comprising: a rotatable chamber having an opening; and a cylindrical intraliposome droplet generating vessel having one or more through holes on its side, the vessel being installed so that the center of a circle of the cross section of the cylinder coincides with the rotation axis of the chamber and rotating integrally with the chamber. (7) The apparatus for producing liposomes according to (6), wherein the chamber and the intra-liposome droplet generating vessel are connected to a rotor of a spin coater and rotated. (8) A liposome droplet generating vessel to be installed in the liposome production apparatus described in (7). [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is a schematic explanatory diagram illustrating a method for producing liposomes by an interface passing method. [Diagram 2] FIG. 2 is a schematic diagram illustrating a method for producing liposomes by the cDICE method. [Diagram 3] FIG. 1 shows a perspective view (right) of one embodiment of a liposome-producing apparatus of the present invention, and an exploded perspective view (left) thereof. [Figure 4] FIG. 2 is a perspective view of an intraliposome droplet generating container (insert) used in the liposome-producing apparatus of the present invention. [Diagram 5] FIG. 2 is a diagram showing the distribution of fluorescence intensity of liposomes produced in an example of the present invention and their cross sections. [Figure 6] 1 is a histogram of the diameters of liposomes produced in the examples of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The cDICE method is a type of interface passing method, so first, the interface passing method will be explained with reference to FIG.

[0011] First, as shown in FIG. 1(a), a liposome external liquid (hereinafter simply referred to as "external liquid") 10 and a lipid liquid 12 are prepared. The external liquid 10 is an aqueous liquid that serves as a medium for suspending liposomes when the liposomes are formed. The lipid liquid 12 is an oily liquid in which liposome-forming lipids 14 are dispersed. The liposome-forming lipids 14 are lipids that have a hydrophilic region and a hydrophobic region in one molecule. The hydrophilic region is usually a region such as phosphocholine, and the hydrophobic region is usually two linear long-chain hydrocarbon groups.

[0012] Next, as shown in FIG. 1B, the external liquid 10 and the lipid liquid 12 are brought into contact with each other, and a droplet 16 of the liposome internal liquid (hereinafter, simply referred to as the "internal liquid") is added to the lipid liquid 12. The internal liquid is an aqueous liquid that is encapsulated in the liposome when the liposome is formed. Then, at the interface between the external liquid 10 and the lipid liquid 12, the lipid molecules 14 start to line up with their hydrophilic regions facing toward the external liquid 10 and their hydrophobic regions facing toward the lipid liquid 12. Similarly, at the interface between the internal droplet 16 and the lipid liquid 12, the lipid molecules 14 start to line up with their hydrophilic regions facing toward the internal droplet and their hydrophobic regions facing toward the lipid liquid 12.

[0013] 1(c), a monolayer 18 is formed at the interface between the external liquid 10 and the lipid liquid 12, in which the hydrophilic regions of the lipid molecules 14 are aligned with their hydrophilic regions facing toward the liposome external liquid 10 and their hydrophobic regions facing toward the lipid liquid 12. Similarly, a monolayer 20 is formed at the interface between the internal droplet 16 and the lipid liquid 12, in which the hydrophilic regions of the lipid molecules 14 are aligned with their hydrophobic regions facing toward the internal droplet 16 and their hydrophobic regions facing toward the lipid liquid 12, and the lipid liquid 12 becomes an emulsion.

[0014] Next, a force such as centrifugal force is applied to the inner droplet 16, causing the inner droplet 16 covered with the lipid monolayer 20 to move towards the outer liquid 10. Eventually, the inner droplet 16 covered with the lipid monolayer 20 collides with the monolayer 18 formed at the interface between the outer liquid 10 and the lipid liquid 12 (see FIG. 1(d)).

[0015] When centrifugal force or other force is continued to be applied to the inner droplet 16, the monolayer 18 at the interface between the external liquid 10 and the lipid liquid 12 and the monolayer 20 at the interface between the internal liquid 16 and the lipid liquid 12 are laminated with the hydrophobic regions of the lipid molecules 14 facing each other to form a lipid bilayer, and a liposome 22 covered with the lipid bilayer is formed (see (e) of Figure 1).

[0016] When centrifugal force or other force is continued to be applied to the inner droplet 16, the formed liposome 22 completely detaches from the monolayer 18, and a liposome 22 covered with a lipid bilayer membrane is formed, floating in the external liquid 10 (see FIG. 1(f)).

[0017] The cDICE method is a type of the above-mentioned interface passing method, and so the principle is the same. The cDICE method is a method in which the above-mentioned interface passing method is performed in a rotating doughnut-shaped chamber, and the interface passing method is performed by applying centrifugal force to the droplets inside by rotating the chamber.

[0018] The cDICE method of the present invention will be described below with reference to Fig. 2. Fig. 2 is a schematic cross-sectional view of a doughnut-shaped chamber 24 for explaining the cDICE method of the present invention. Note that the scale of each component has been changed for ease of understanding, and is significantly different from the scale of each component in the actual method. Also, components similar to those in the interface passing method described above with reference to Fig. 1 are given the same reference numbers as in Fig. 1.

[0019] The rotatable doughnut-shaped chamber 24 itself is known, and is arranged such that the rotation axis is vertical and the diameter of the doughnut extends in a direction perpendicular to the rotation axis (horizontal direction), and rotates within a horizontal plane. The chamber 24 can be rotatable by being connected to the rotor of the spin coater so that the rotation axis of the chamber 24 is coaxial with the rotation axis of the rotor of the spin coater, but is not necessarily limited to this.

[0020] In the cDICE method of the present invention, first, an external liquid layer 10 is formed in the outer region of the chamber 24. Here, the "outer region" means the side farther from the rotation axis of the chamber 24. When the chamber 24 is stationary and an external liquid is injected into the chamber 24 through a lidded opening provided in the chamber 24, and then the chamber 24 is rotated, the external liquid moves to the outer region of the chamber 24 by centrifugal force and is pressed against the side wall of the chamber 24 to form the external liquid layer 10. The external liquid itself is well known and is an aqueous liquid as described above, and usually, an aqueous solution of a water-soluble substance such as glucose is used to obtain a desired osmotic pressure. The concentration of the water-soluble substance such as glucose can be set appropriately, but is usually about 50 mM to 200 mM. The thickness of the external liquid layer 10 is not particularly limited and is set appropriately, but usually, the radial length of the chamber 24 is the thickness when about 1 mL of external liquid is poured into the chamber 24 using a pipette or the like. The rotation speed of the chamber is set appropriately, and the acceleration at the interface between the external liquid layer 10 and the lipid liquid layer 12 can be set to, for example, about 500×g to 1500×g.

[0021] Next, while the chamber 24 is rotating, the lipid liquid 12 is supplied to the center of the doughnut. Then, the lipid liquid 12 moves radially outward of the chamber 24 by centrifugal force and is pressed against the external liquid layer 10, forming a lipid liquid layer 12 in contact with the external liquid layer 10 in the inner region of the chamber 24. Here, the "inner region" means a region that is radially inward from the outer region described above. The operation of supplying the lipid liquid to the center of the doughnut can be easily performed by supplying the lipid liquid to a later-described intraliposome droplet generating container that is inserted into the doughnut portion. As described above, the lipid liquid is an oily liquid containing a liposome-forming lipid. Liposome-forming lipids are well known, and phospholipids in which two linear long-chain hydrocarbon groups and one hydrophilic site such as phosphocholine or phosphoethanolamine are bound to glycerin are widely used. The carbon number of the long chain hydrocarbon is usually about 16 to 20, and for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is widely used. The concentration of the liposome-forming lipid in the lipid solution 12 is not particularly limited and may be appropriately set, but is usually about 0.5 mM to 20 mM.

[0022] Next, while the chamber 24 is rotating, the inner droplets 16 are supplied to the lipid liquid layer 12. This method of supplying the inner droplets 16 is an important feature of the present invention. In the method of the present invention, the inner liquid is supplied near the rotation axis of the chamber 24, i.e., the center part of the donut, and the inner liquid is passed through one or more through holes by centrifugal force to supply the inner droplets to the lipid liquid layer. Although the number of through holes may be one, it is preferable to provide multiple through holes because the production efficiency of liposomes is higher. The inner liquid is an aqueous liquid to be encapsulated inside the liposome. Basically, an aqueous solution similar to the above-mentioned external liquid is used, but an aqueous solution in which the target substance to be encapsulated inside the liposome is dissolved is usually used.

[0023] This operation can be easily performed by using a cylindrical intraliposomal droplet generating vessel having one or more through holes on the side surface. That is, a cylindrical intraliposomal droplet generating vessel having one or more through holes on the side surface is installed so that the center of the circle of the cross section of the cylinder coincides with the rotation axis of the chamber 24, and the internal liquid is placed in the intraliposomal droplet generating vessel while the chamber 24 and the intraliposomal droplet generating vessel are rotating integrally. Then, the internal liquid passes through the through holes on the side surface of the intraliposomal droplet generating vessel by centrifugal force to become internal droplets, and moves into the lipid liquid layer. In this way, the internal droplets 16 are supplied to the lipid liquid layer 12 by centrifugal force.

[0024] The intraliposomal droplet generating vessel can be prepared by providing an opening on the side of a cylinder and covering the opening with the membrane having the through-hole. In this case, the size of the opening is not limited and can be set appropriately, for example, the width can be about 10% to 40% of the circumference, particularly 20% to 30%, and the height can be about 1 mm to 5 mm, particularly 2 mm to 3 mm. The membrane for covering such an opening is not limited in any way as long as it has a minute through-hole, but a track-etched membrane (also called a "track-etched membrane") is preferable. A track-etched membrane is a membrane produced by irradiating a resin film such as a polycarbonate film or a polyester film with gamma rays to open minute holes called tracks, and then chemically etching the film to expand the tracks to form through-holes. The diameter and density of the through-holes can be accurately controlled, and through-holes with uniform diameters are clearly formed in a thin film of uniform thickness. Track-etched membranes are commercially available in various hole sizes, so commercially available products can be advantageously used.

[0025] The above-mentioned intraliposome droplet generating container can be attached to the chamber 24 as described below, and is preferable for easy handling, but is not essential. For example, by providing a through hole in the inner wall (the side closer to the center of rotation) of the doughnut-shaped chamber, it is possible to supply the intraliposome droplets 16 to the lipid liquid layer 12 through the through hole by centrifugal force.

[0026] The pore size (diameter) of the through hole for generating the inner droplet 16 can be appropriately set according to the diameter of the target liposome. In the following example, when liposomes were produced using a track-etched membrane with a pore size of 14 μm, liposomes with an average diameter of about 10 μm were produced.

[0027] Next, a preferred embodiment of a liposome-producing apparatus for the cDICE method for carrying out the above-mentioned method of the present invention will be described.

[0028] The right side of FIG. 3 is a schematic perspective view of a preferred embodiment of the liposome-producing apparatus of the present invention, and the left side of FIG. 3 is an exploded perspective view thereof.

[0029] As shown in the right diagram of FIG. 3, a liposome-producing apparatus which is a preferred embodiment of the liposome-producing apparatus of the present invention includes a rotatable doughnut-shaped chamber 24. A rotating shaft 28 connected to a spin coater is provided at the bottom of the chamber 24. By connecting the rotating shaft 28 to the rotating shaft of the spin coater, the chamber 24 rotates together with the rotation of the rotor of the spin coater. An intra-liposome droplet generating container 30 is inserted in the center of the chamber 24. Since the intra-liposome droplet generating container 30 is inserted and installed in the cavity at the center of the doughnut, it may be referred to as an "insert" for convenience in the following description and examples.

[0030] The left side of Fig. 3 is an exploded perspective view of the liposome-producing apparatus shown on the right side. The chamber 24 is composed of a thin cylindrical chamber body 24a and a doughnut-shaped chamber lid 24b that is placed on the top of the outer peripheral wall of the chamber body 24a and covers the chamber body 24a. The chamber lid 24b is provided with two openings 26 for injecting an external liquid or recovering the produced liposome-containing liquid. The area surrounded by the outer peripheral wall of the chamber body 24a forms a space. The bottom surface of the chamber body 24a is provided with a shallow recess 24c for receiving the insert 30 and a cross-shaped protrusion 24d for fitting into the bottom of the insert 30.

[0031] The insert 30 is inserted into the central cavity of the doughnut-shaped chamber lid 24b. The top of the insert 30 is covered with a doughnut-shaped insert lid 32 having an opening in the center. A cross-shaped recess (not shown) that fits with the cross-shaped protrusion 24d described above is provided on the outside of the bottom surface of the insert 30. The insert 30 is placed in the shallow recess 24c described above, and is fixed in the horizontal direction by fitting the protrusion 24d into the recess (not shown). When the chamber 24 rotates, the insert 30 also rotates integrally therewith.

[0032] A schematic perspective view of the insert 30 is shown in Fig. 4. As shown in Fig. 4, an opening 34 is provided in the lower part of the side wall of the insert 30, and this opening 34 is covered with the above-mentioned track etch film (not shown).

[0033] The liposome-producing apparatus according to this specific example can be easily produced by producing the above-mentioned four components, the chamber body 24a, the chamber lid 24b, the insert 30, and the insert lid 32, using a 3D printer, covering the opening 32 of the insert 30 with a track-etched membrane using an adhesive, and further bonding these together with an adhesive. Note that, although the liposome-producing apparatus is made of resin when produced using a 3D printer, it is also possible to construct the apparatus from metal or from metal and resin.

[0034] The operation of producing liposomes using the liposome-producing apparatus according to the embodiment of the present invention described above can be carried out according to the method of the present invention described above (see FIG. 2).

[0035] First, while the chamber 24 is stationary, the external liquid is injected into the chamber 24 through the opening 26. After the injection, the opening 26 is sealed with adhesive tape. In this state, the chamber 24 is rotated, and the external liquid is moved to the outer region inside the chamber 24 by centrifugal force, forming the external liquid layer 10.

[0036] Next, lipid liquid is supplied into the insert 30 from the top of the insert 30 through the central opening of the insert lid 32. The lipid liquid then enters the chamber 24 through the through holes in the track-etched membrane due to centrifugal force, and is further moved outward by centrifugal force to form the lipid liquid layer 12.

[0037] Next, while continuing the rotation, the internal liquid is supplied from the top of the insert 30 through the central opening of the insert lid 32. Then, the internal liquid enters the chamber 24 through the through holes of the track-etched membrane by centrifugal force. At this time, the internal liquid is cut by the through holes to limit the size of the internal liquid, and the internal liquid entering the chamber 24 is supplied into the lipid liquid layer 12 as droplets smaller than the diameter of the through holes. Here, the internal liquid supplied to the insert 30 may be the internal liquid alone, but it is preferable to dilute the internal liquid with the lipid liquid in order to appropriately control the production rate of liposomes. At this time, the volume of the lipid liquid added to the internal liquid is the volume of the part of the space between the chamber 24 and the insert 30 that is not occupied by the external liquid 10 and the lipid liquid 12.

[0038] As the rotation continues, the inner droplet 16 passes through the through holes in the track-etched membrane and moves outward due to centrifugal force. During the movement, a monolayer of lipid molecules is formed on the surface of the inner droplet 16, as described above. As the rotation continues, the inner droplet 16 covered with the monolayer reaches the monolayer formed at the interface between the outer liquid layer 10 and the lipid liquid layer 12, breaks through it, and a liposome covered with a lipid bilayer is formed in the outer liquid layer 10.

[0039] The generated liposomes can be collected as follows. The rotation of the chamber 24 is stopped, the chamber 24 is removed from the spin coater, the adhesive tape on one of the openings 26 is peeled off, and the chamber 24 is slowly tilted in a direction in which the opening is on the lower side. Then, the aqueous external liquid layer 10 is on the lower side, and the lipid liquid layer 12 is on the upper side. In this state, a pipette is inserted into the aqueous external liquid layer 10 from the opening 26, and the external liquid layer 10 containing the generated liposomes is collected. The collected external liquid layer 10 is then placed in a centrifuge tube, and centrifuged at an acceleration of 1000×g for about 3 to 5 minutes to precipitate the external liquid layer, and the supernatant liquid (containing the mixed lipid liquid and excess external liquid) is removed with a pipette, and the liposomes precipitated at the bottom of the centrifuge tube can be collected.

[0040] The present invention will be described in detail below with reference to examples, although the present invention is not limited to the following examples.

[0041] Example 1: Preparation of a liposome production device The liposome-producing device described based on FIG. 3 and FIG. 4 was produced. The four components of the chamber body 24a, the chamber lid 24b, the insert 30, and the insert lid 32 were each produced by a 3D printer. The track-etched membrane was attached to the opening 32 of the insert 30 with an acrylic adhesive to cover it. These four components were assembled. That is, the insert 30 was placed in the shallow recess 24c of the chamber body 24a, and the protrusion 24d was fitted with a recess (not shown) provided on the outside of the bottom of the insert 30 to fix the insert 30 in the horizontal direction. Furthermore, these were bonded with an acrylic adhesive to produce the liposome-producing device of the present invention. As the track-etched membrane, a commercially available product (manufactured by GVS) in which a through hole with a diameter of 14 μm was opened in a polycarbonate film was used. The inner diameter of the chamber 24 was 46 mm, and the height was 3 mm. The inner diameter of the insert 30 was 12 mm, the distance from the central axis of rotation to the track-etched film was 8 mm, and the width of the opening 34 covered by the track-etched film was approximately 12 mm along the side of the insert 30, and the height was 2 mm.

[0042] Example 2 Preparation of liposomes Liposomes were produced using the liposome production device prepared in 1 above. The following operations were performed. The liposome production device was fixed to the rotation axis of a spin coater. 1.22 mL of external solution was filled into the chamber 24 from one opening 26 of the chamber lid 24b, and the two openings 26 were sealed with adhesive tape (Kapton Tape (trade name)). The external solution was a 100 mM glucose aqueous solution. The device was rotated at 6200 rpm. At this time, a force of about 340×g was applied at the position of the track etch membrane 8 mm from the rotation axis, and a force of about 860×g was applied at the position of the interface between the external solution and the lipid solution to be injected in the next step from the rotation axis. While rotating, 2.83 mL of lipid solution was injected into the inside of the insert 30 from the opening of the insert lid 32. The lipids were DOPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)ammonium salt (RhPE), which was prepared by dispersing 2.5 mM DOPC and 0.1 mol% RhPE in mineral oil in 10 wt% chloroform. RhPE is a red lipid and was used for subsequent microscopic observations.

[0043] On the other hand, 250 μL of the lipid solution was placed in a tube, and 10 μL of the internal solution was added thereto. The internal solution was 100 mM sucrose and 10 μM dextran (Alexa Flour 488-dextran 3k The dextran used contained a green fluorescent substance and was used for microscopic observation after the formation of liposomes. The entire amount of this aqueous solution was taken with a pipette and injected into the inside of the insert 30 through the opening of the insert lid 32.

[0044] After 5 minutes, the rotation was stopped and the device was removed from the spin coater. The adhesive tape sealing one of the openings 26 was peeled off, the chamber 24 was slowly tilted, and the aqueous phase in the chamber 24 was gently collected near the opening where the adhesive tape had been peeled off. A pipette was inserted into the aqueous phase in the chamber through the opened opening, and the aqueous phase was slowly aspirated and collected in a microtube. The collected liposome solution was centrifuged at 1,000 x g for 3 to 5 minutes to remove any lipid dispersion oil that had been mixed in. The lipid dispersion oil and excess supernatant were carefully removed with a pipette, and the liposomes that had settled at the bottom of the tube were collected.

[0045] Example 3 Analysis of formed liposomes The liposomes formed in Example 2 were sealed in a glass chamber coated with bovine serum albumin (BSA) and fluorescence observation was performed using a confocal microscope. Insets show plots of red and green fluorescence intensity along the dashed lines. The results are shown in Figure 5.

[0046] There is a broad green fluorescence peak between the two red fluorescence peaks corresponding to the liposome membrane, suggesting that a green fluorescent substance is encapsulated in the liposome. In addition, as can be seen in the spectrum cross section in the inset of Figure 5, the liposome internal liquid containing the green fluorescent substance is sandwiched between lipid membranes containing red fluorescent lipids. This indicates that liposomes encapsulating green fluorescent substances have been formed.

[0047] In addition, the equivalent diameter was calculated from the area of ​​each liposome in the image, and a histogram was drawn. The results are shown in Figure 6. As shown in Figure 6, it was found that liposomes with a diameter approximately equal to or smaller than the pore diameter (14 μm) of the track-etched membrane used were formed. The average liposome diameter was 9.7 ± 2.7 μm (mean ± standard deviation). [Explanation of symbols]

[0048] 10 External fluid (layer) 12 Lipid liquid (layer) 14 Lipid molecules 16 Internal fluid (drops) 18 Monolayer 20 Monolayer 22 Liposomes 24 Chambers 24a Chamber body 24b Chamber lid 24c Shallow Recess 24d Convex 26 Aperture 28 Rotational Axis 30 Liposome droplet generation vessel (insert) 32 Insert Lid 34 Opening

Claims

1. forming an outer liposomal liquid layer in an outer region within a rotatable chamber; forming a lipid liquid layer in contact with the liposome external liquid layer in an inner region of the chamber; A method for producing liposomes by a cDICE method, comprising the steps of: supplying intraliposomal droplets to the lipid liquid layer while rotating the chamber; and passing the intraliposomal droplets through an interface between the liposomal external liquid layer and the lipid liquid layer by centrifugal force, thereby generating liposomes surrounded by a lipid bilayer membrane in the liposomal external liquid layer, A method for producing liposomes, comprising the steps of supplying liposome internal liquid near a rotation axis of the chamber, causing the liposome internal liquid to pass through one or more holes by centrifugal force to supply liposome internal liquid droplets to the lipid liquid layer, and moving the supplied liposome internal liquid droplets further outward by centrifugal force to pass through the interface.

2. The method according to claim 1, wherein a cylindrical intraliposomal droplet generating vessel having one or more through holes on a side surface is installed so that a center of a circle of a cross section of the cylinder coincides with the rotation axis, and the intraliposomal liquid is introduced into the intraliposomal droplet generating vessel while the chamber is rotating, and the intraliposomal droplets are supplied to the lipid liquid layer by passing through the through holes on the side surface of the intraliposomal droplet generating vessel.

3. 3. The method according to claim 2, wherein the container has an opening extending through a side surface thereof, the opening being covered by the membrane having the through hole.

4. 4. The method of claim 3, wherein said membrane is a track-etched membrane having a plurality of said through holes.

5. The method of claim 4, wherein the through holes have a diameter of 1 μm to 20 μm.

6. A liposome production apparatus for the cDICE method, comprising: a rotatable chamber having an opening; and a cylindrical intraliposome droplet generating vessel having one or more through holes on its side, the vessel being installed so that the center of a circle of the cross section of the cylinder coincides with the rotation axis of the chamber and rotating integrally with the chamber.

7. 7. The liposome-producing apparatus according to claim 6, wherein the chamber and the intra-liposome droplet generating vessel are connected to a rotor of a spin coater and rotated.

8. A liposome droplet generating vessel, which is installed in the liposome production apparatus according to claim 7.

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