JP2018086630A - Fixing implement and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an improved liposome-forming technology.SOLUTION: A fixing implement includes an outer pipe sending a first droplet material and an inner pipe disposed inside the outer pipe and sending a second droplet material, and fixes the inner pipe to the inside of the outer pipe of a droplet producing device where fluid-sending directions of the first droplet material and the second droplet material are the same, and the tip end part of the inner pipe on the downstream side in the fluid-sending direction is located on an upstream side in the fluid-sending direction of the tip end part of the outer pipe on the downstream side in the fluid-sending direction, and is provided with an outer pipe holding member holding the outer pipe, an inner pipe holding member holding the inner pipe, and a connection member connecting the outer pipe holding member and the inner pipe holding member. The connection member holds the outer pipe with the outer pipe holding member, holds the inner pipe with the inner pipe holding member and connects the pipes so that the distance between the outer pipe holding member and the inner pipe holding member in the fluid-sending direction can be adjusted while the inner pipe is disposed inside the outer pipe.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a fixture and its use. More specifically, the present invention relates to a fixture, a method for producing a droplet, and a method for adjusting the particle size of the droplet.

  Liposomes are composed of lipid bilayers and are artificially produced closed vesicles. Since it can have the same structure as a cell, it is highly safe even when administered to a living body. For this reason, the technique which uses the liposome which included the reagent as a pharmaceutical or cosmetics is implement | achieved. In addition, it is also used for the purpose of investigating the function of a cell by preparing a liposome imitating a cell by putting a protein or the like inside the liposome at a high density.

  As a method for encapsulating a reagent in a liposome using a simple instrument in a laboratory or the like, there are a hydration method and an extruder method. However, these methods have a low efficiency of encapsulating reagents in liposomes and require a large amount of reagents, and are therefore very inefficient when encapsulating expensive or rare reagents.

  On the other hand, Patent Document 1 and Non-Patent Document 1 describe a method in which a reagent can be encapsulated in liposomes more efficiently than a hydration method or an extruder method.

International Publication No. 2015/137380

Yamada, A. et al, Spontaneous Transfer of Phospholipid-Coated Oil-in-Oil and Water-in-Oil Micro-Droplets through an Oil / Water Interface, Langmuir, 22 (24), 9824-9828, 2006.

  However, in the method of Patent Document 1, the number of liposomes that can be produced tends to be small. Also, depending on the type of reagent, it may be difficult to encapsulate the liposome. Moreover, in the method described in Non-Patent Document 1, the size of the obtained liposome is not uniform. In addition, since there is a step of preparing an emulsion by stirring, it may be denatured if the reagent to be included is unstable with respect to the oil layer. In view of such a background, an object of the present invention is to provide an improved liposome forming technique.

The present invention includes the following aspects.
[1] An outer tube that feeds the first droplet material and an inner tube that is arranged inside the outer tube and feeds the second droplet material, and the outer tube and the inner tube There is a gap between the first droplet material and the second droplet material, and the tip of the inner tube on the downstream side in the liquid feeding direction is the outer portion. A fixture for fixing the inner tube to the inside of the outer tube of a droplet manufacturing device located on the upstream side in the liquid feeding direction with respect to the tip portion on the downstream side in the liquid feeding direction of the tube, An outer tube holding member for holding, an inner tube holding member for holding the inner tube, and a connecting member for connecting the outer tube holding member and the inner tube holding member, the connecting member comprising the outer tube The outer tube holding member is held by the holding member, the inner tube is held by the inner tube holding member, and the inner tube is disposed inside the outer tube. Adjustably connecting the distance of said feeding direction of the inner tube holding member, fixture.
[2] The fixture according to [1], which is made of metal.
[3] An outer tube that feeds the first droplet material and an inner tube that is arranged inside the outer tube and feeds the second droplet material, and the outer tube and the inner tube There is a gap between the first droplet material and the second droplet material, and the tip of the inner tube on the downstream side in the liquid feeding direction is the outer portion. The first droplet material is fed to the outer tube of the droplet manufacturing device located on the upstream side in the liquid feeding direction with respect to the tip of the tube on the downstream side in the liquid feeding direction, and the first droplet material is fed to the inner tube. A step of delivering a liquid droplet material, wherein the liquid droplet is a liposome, the first liquid droplet material includes a lipid constituting a membrane of the liposome, and the second liquid droplet material is highly hydrophilic. A method for producing droplets comprising molecules.
[4] The method for producing droplets according to [3], wherein the hydrophilic polymer is a polysaccharide or a polyether.
[5] An outer tube that feeds the first droplet material and an inner tube that is arranged inside the outer tube and feeds the second droplet material, and the outer tube and the inner tube There is a gap between the first droplet material and the second droplet material, and the tip of the inner tube on the downstream side in the liquid feeding direction is the outer portion. The first droplet material is fed to the outer tube of the droplet manufacturing device located on the upstream side in the liquid feeding direction with respect to the tip of the tube on the downstream side in the liquid feeding direction, and the first droplet material is fed to the inner tube. Including a step of feeding the droplet material of 2, wherein the feeding is performed by centrifuging the device, and adjusting the centrifugal force applied to the device to adjust the particle size of the droplet to be manufactured. A method of adjusting the droplet size.

  According to the present invention, an improved liposome forming technique can be provided.

It is a schematic diagram explaining the structure of an example of a fixing tool and the droplet manufacturing device assembled using the said fixing tool. (A) is the front view, side view, and top view of an inner pipe holding member. (B) is the front view, side view, top view, and bottom view of an outer tube holding member. It is a schematic diagram explaining the structure of an example of a conventional fixture. (A)-(c) is a schematic diagram explaining an example of the procedure which arrange | positions an outer tube | pipe and an inner tube | pipe concentrically using a conventional fixture. It is a schematic diagram explaining the manufacturing method of a droplet. It is a schematic diagram which shows a mode that the liposome which included the reagent is manufactured. (A)-(d) are the microscope pictures of the liposome produced in Experimental example 1. FIG. (A) And (b) is the microscope picture of the liposome produced in Experimental example 2. FIG. (A) And (b) is a graph which shows the measurement result of the particle size and presence rate of the liposome produced in Experimental example 3. FIG.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant description is omitted. Note that the dimensional ratio in each drawing is exaggerated for the sake of explanation, and does not necessarily match the actual dimensional ratio.

[Fixture]
In one embodiment, the present invention includes an outer tube that feeds a first droplet material, and an inner tube that is disposed inside the outer tube and feeds a second droplet material. There is a gap between the tube and the inner tube, the liquid feeding direction of the first droplet material and the second droplet material is the same, and the downstream side of the liquid feeding direction of the inner tube It is a fixture for fixing the inner tube to the inside of the outer tube of the liquid droplet manufacturing device whose tip is located upstream of the tip of the outer tube on the downstream side in the liquid feeding direction. An outer tube holding member that holds the outer tube, an inner tube holding member that holds the inner tube, and a connecting member that connects the outer tube holding member and the inner tube holding member. The member holds the outer tube by the outer tube holding member, holds the inner tube by the inner tube holding member, and arranges the inner tube inside the outer tube. In state, adjustably connecting the distance feeding direction between the inner tube holding member and the outer tube holding member, providing a fixture.

  FIG. 1 is a schematic diagram illustrating an example of a fixture of the present embodiment and the structure of a droplet manufacturing device assembled using the fixture. A front view and a plan view are shown.

  The droplet manufacturing device 100 includes an outer tube 10 and an inner tube 20. A gap exists between the outer tube 10 and the inner tube 20. Further, the distal end portion 22 on the downstream side in the liquid feeding direction of the inner tube 20 is located upstream of the distal end portion 12 on the downstream side in the liquid feeding direction of the outer tube 10.

  In the droplet manufacturing device 100, the outer tube 10 and the inner tube 20 are fixed by the fixture 40 in a state where the inner tube 20 is disposed inside the outer tube 10. The fixture 40 includes an outer tube holding member 11, an inner tube holding member 21, and a connecting member 30. The connecting member 30 holds the outer tube 10 with the outer tube holding member 11, holds the inner tube 20 with the inner tube holding member 21, and places the inner tube 20 inside the outer tube 10, with the outer tube holding member 11 and the inner tube holding member 21 are connected so that the distance in the liquid feeding direction can be adjusted. The connecting member 30 includes a pin 31, a fixing screw 32, and a fixing screw 33.

  FIG. 2A is a front view, a side view, and a plan view of the inner tube holding member 21. FIG. 2B is a front view, a side view, a plan view, and a bottom view of the outer tube holding member 11.

  The outer tube holding member 11 and the inner tube holding member 21 connected by the connecting member 30 are movable along the pins 31 to arbitrary positions. Further, the inner tube holding member 21 can be fixed on the pin 31 by tightening the fixing screw 32. Further, the outer tube holding member 11 can be fixed on the pin 31 by tightening the fixing screw 33.

  Here, a conventional fixture will be described. FIG. 3 is a schematic diagram illustrating the structure of an example of a conventional fixture described in Patent Document 1. As shown in FIG. The conventional fixture 40 ′ is a fixture used to arrange the inner tube 20 constituting the droplet manufacturing device inside the outer tube 10, and is an outer tube holding member 11 that holds the outer tube 10. And an inner tube holding member 21 that holds the inner tube 20.

  The outer tube holding member 11 and the inner tube holding member 21 are plate-like members having a hole in the center, and the outer tube holding member 11 and the inner tube holding member 21 are connected by a connecting member 30 '. The connecting member 30 ′ is a screw that rotatably connects the outer tube holding member 11 and the inner tube holding member 21. The fixture 40 ′ further includes supports 11 ′ and 21 ′, which are plate-like members having a hole in the center, in order to support the outer tube 10 and the inner tube 20 and to maintain a concentric arrangement more effectively. ing.

  Subsequently, an example of a procedure for arranging the outer tube 10 and the inner tube 20 concentrically using the conventional fixture 40 ′ will be described with reference to FIGS. 4 (a) to 4 (c). In the fixture 40 ′ shown in FIG. 4A, the holes 11a, 11′a, 21a and 21′a of the outer tube holding member 11, the support 11 ′, the inner tube holding member 21 and the support 21 ′ are concentric. Arranged in a shape.

  First, as shown in FIG. 4B, the inner tube holding member 21 and the support 21 ′ are rotated, the centers of the holes of the outer tube holding member 11 and the support 11 ′, the inner tube holding member 21 and The outer tube 10 is inserted into each hole of the outer tube holding member 11 and the support member 11 ′ by shifting the center of each hole of the support member 21 ′.

  Here, since the outer diameter of the outer tube 10 is larger toward the upstream side in the fluid feeding direction, the outer tube 10 is fitted into the outer tube 10 by passing the outer tube 10 through the hole 11 a provided in the outer tube holding member 11. The tube 10 is held.

  Next, as shown in FIG. 4 (c), the inner tube holding member 21 and the support 21 ′ are rotated, the centers of the holes of the outer tube holding member 11 and the support 11 ′, the inner tube holding member 21 and The inner tube 20 is inserted into each hole of the inner tube holding member 21 and the support body 21 ′ by matching the center of each hole of the support body 21 ′.

  Here, since the outer diameter of the inner tube 20 is larger toward the upstream side in the fluid feeding direction, the inner tube 20 is fitted by passing the inner tube 20 through a hole 21 a provided in the inner tube holding member 21. 20 is held. In this way, the outer tube 10 and the inner tube 20 can be arranged concentrically.

  In the conventional fixture 40 ′, the outer tube holding member 11, the support 11 ′, the inner tube holding member 21, and the support 21 ′ are connected by a connecting member 30 ′ that is a screw. For this reason, the distance between the outer tube holding member 11 and the inner tube holding member 21 can be adjusted only by a multiple of the distance between the threads of the connecting member 30 ′. Moreover, since it is necessary to arrange the outer tube 10 and the inner tube 20 after adjusting the distance between the outer tube holding member 11 and the inner tube holding member 21, the outer tube holding member 11 and the inner tube are observed while observing under a microscope. The distance from the holding member 21 could not be adjusted.

  On the other hand, according to the fixture 40 of the present embodiment, after the inner tube 20 of the droplet manufacturing device 100 is temporarily fixed inside the outer tube 10, the distal end portion 22 of the inner tube 20 and the outer tube 10 are fixed. While observing the distal end portion 12 under a microscope, the distance in the liquid feeding direction between the outer tube holding member 11 and the inner tube holding member 21 can be adjusted. Further, the distance between the outer tube holding member 11 and the inner tube holding member 21 can be adjusted steplessly.

  For this reason, as will be described later in the embodiment, the inner tube 20 of the droplet manufacturing device 100 can be arranged and fixed at an optimal position inside the outer tube 10, and the manufacturing efficiency of the droplets can be dramatically increased. be able to.

  The fixture of the present embodiment may be made of resin or metal. Especially, it is preferable that it is metal. That the fixture of this embodiment is made of metal may be that at least a part of the fixture is made of metal, or may be made entirely of metal, but is made entirely of metal. It is preferable that

  Examples of the metal include aluminum, stainless steel, and titanium. Since metal can be easily finely processed, it is easy to manufacture a highly accurate fixture by using a metal material. In addition, since the metal tends to have less fluctuation with respect to temperature and pressure than the resin, it is easy to always fix the outer tube and the inner tube at the correct positions.

  In addition, in the droplet manufacturing device, when the droplet material is sent by centrifugation, the fixture needs to withstand centrifugal force. On the other hand, by using a metal material, a fixture that can withstand centrifugal force can be easily manufactured.

  Then, an example of the procedure which arrange | positions the outer tube | pipe 10 and the inner tube | pipe 20 concentrically using the fixing tool 40 of this embodiment is demonstrated. In the fixture 40 shown in FIG. 1, the hole of the outer tube holding member 11 and the hole of the inner tube holding member 21 are arranged concentrically.

  First, the inner tube holding member 21 is rotated to shift the center of the hole of the outer tube holding member 11 from the center of the hole of the inner tube holding member 21, and the outer tube 10 is inserted into the hole of the outer tube holding member 11. insert.

  Here, since the outer diameter of the outer tube 10 is larger toward the upstream side in the fluid feeding direction, the outer tube 10 is fitted by being passed through a hole provided in the outer tube holding member 11, and the outer tube 10 is held. .

  Next, the inner tube holding member 21 is rotated so that the center of the hole of the outer tube holding member 11 and the center of the hole of the inner tube holding member 21 coincide with each other, and the inner tube 20 is aligned with the hole of the inner tube fixing member 21. Insert.

  At this time, in order to surely align the center, the workability is improved if the subsequent work is performed after being set on a center alignment holder that covers the fixture 40.

  Here, since the outer diameter of the inner tube 20 is larger toward the upstream side in the fluid feeding direction, the inner tube 20 is fitted and held by passing the inner tube 20 through a hole of the inner tube holding member 21. Is done. In this way, the outer tube 10 and the inner tube 20 can be arranged concentrically.

  In the fixture 40, the outer tube holding member 11 and the inner tube holding member 21 are connected by the pin 31 of the connecting member 30. Therefore, each member can be shifted and the distance between members can be adjusted with the holder fitted. By adjusting the position while observing the position of the distal end portion 12 of the inner tube 20 and the inner diameter of the outer tube 10 with a microscope or the like, the overlapping position of the outer tube 10 and the inner tube 20 can be adjusted appropriately.

  After the positions of the outer tube 10 and the inner tube 20 are adjusted to the optimum positions, it is necessary to fix the positions of the respective members by tightening the fixing screws 32 and 33. One of the fixing screw 32 or the fixing screw 33 may be always fastened and fixed, or may be fixed with an adhesive or the like.

[Droplet production method]
In one embodiment, the present invention includes an outer tube that feeds a first droplet material, and an inner tube that is disposed inside the outer tube and feeds a second droplet material. There is a gap between the tube and the inner tube, the liquid feeding direction of the first droplet material and the second droplet material is the same, and the downstream side of the liquid feeding direction of the inner tube The first droplet material is fed to the outer tube of the droplet manufacturing device located at the upstream side in the liquid feeding direction with respect to the tip portion of the outer tube on the downstream side in the liquid feeding direction, A step of feeding the second droplet material to the inner tube, wherein the droplet is a liposome, the first droplet material includes a lipid constituting a membrane of the liposome, and the second liquid A method for producing droplets is provided wherein the droplet material comprises a hydrophilic polymer.

  A method for producing droplets according to this embodiment will be described. FIG. 5 is a schematic diagram for explaining a method for producing droplets according to the present embodiment. In FIG. 5, the above-described droplet manufacturing device 100 is accommodated in the 1.5 mL tube. The outer tube 10 and the inner tube 20 of the droplet manufacturing device 100 are preferably fixed by the fixture 40 described above.

  Inside the 1.5 mL tube, there is prepared a lipid membrane M1 formed by a liquid feeding operation, which will be described later, through which a droplet of one lipid membrane passes. Specifically, the water phase W1, the oil phase O1, and the lipid membrane M1 formed at the interface between the water phase W1 and the oil phase O1 are formed inside the 1.5 mL tube. The liquid level of the oil layer O1 and the distal end portion 12 of the outer tube 10 are separated. The distance between the liquid surface of the oil layer O1 and the distal end portion 12 of the outer tube 10 may be, for example, 1 to 20 mm or 1 to 10 mm.

  Here, the composition of the oil phase O1 may be the same as or different from the composition of the first droplet material 10F described later. Examples of the composition of the aqueous phase W1 include water, an isotonic solution or a buffer solution.

  In the droplet manufacturing device 100, the outer tube 10 and the inner tube 20 are arranged concentrically. The outer pipe 10 and the inner pipe 20 have a perfect cross-sectional shape. As shown in FIG. 5, the inner tube 20 is disposed inside the outer tube 10, and a gap is formed between the outer tube 10 and the inner tube 20.

  The inside (gap) of the outer tube 10 is filled with the first droplet material 10F. The inner tube 20 is filled with a second droplet material 20F. The first droplet material 10F and the second droplet material 20F are fed in the axial direction of the outer tube 10 and the inner tube 20. The liquid feeding directions of the first droplet material 10F and the second droplet material 20F are the same, and are the directions of the arrows in FIG. The distal end portion 22 of the inner tube 20 on the downstream side in the liquid feeding direction is located upstream of the distal end portion 12 of the outer tube 10 on the downstream side in the liquid feeding direction.

  As the first droplet material 10F, an amphiphilic substance constituting a liposome can be used. The amphiphilic substance is preferably dissolved in an organic solvent that is difficult to dissolve in water.

  Examples of amphiphilic substances include lipids and surfactants. Examples of lipids include artificial (synthetic) lipids, lipids derived from natural products, and lipids extracted from cells. Specifically, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and the like. ), Cholesterol, sphingomyelin, glycolipid ganglioside, lecithin and the like. Examples of the surfactant include Span 80, Tween 80, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), and the like. One kind of amphiphilic substance may be used alone, or two or more kinds may be mixed and used.

  Examples of the organic solvent include vegetable oils such as mineral oil and olive oil, silicon oil, decane, hexane, and chloroform.

  The second droplet material 20F includes a hydrophilic polymer. The second droplet material 20F may further include a target substance to be included in the liposome. Here, the target substance itself to be encapsulated in the liposome may be a hydrophilic polymer. As will be described later in Examples, according to the method for producing droplets of this embodiment, the second droplet material contains a hydrophilic polymer, thereby improving the production efficiency of liposomes encapsulating a desired substance. Can be made.

  In the second droplet material 20F, the target substance and the hydrophilic polymer are preferably dissolved or dispersed in water. The substance to be encapsulated in the liposome is not particularly limited, and examples thereof include proteins, nucleic acids, viruses, bacteria, cell extracts, polysaccharides, low molecular compounds, and particles. Examples of the particles include magnetic beads and resin beads.

  In the droplet production method of the present embodiment, examples of the hydrophilic polymer include polysaccharides and polyethers.

  Examples of the polysaccharide include dextran, dextrin, and pectin. The molecular weight of the polysaccharide is preferably 1000 to 300000.

  Examples of the polyether include polyethylene glycol and polypropylene glycol. The molecular weight of the polyether is preferably 1000 to 300,000.

  In the droplet manufacturing method of the present embodiment, the second droplet material preferably further contains an additive for increasing the specific gravity of the second droplet material. This makes it easier to further increase the efficiency of encapsulating the target substance in the liposome.

  The additive is not particularly limited as long as it is a substance that increases the specific gravity, but it is preferable not to use an amphipathic substance or the like that makes the liposome membrane unstable. In addition, when the prepared reagent-encapsulated liposome is used for cell experiments, it is preferably a substance having no cytotoxicity.

  The specific gravity of the second droplet material is preferably adjusted to 1.01 to 1.5. Examples of the additive for increasing the specific gravity include low-molecular sugars and high-density media (for example, iodixanol). Examples of the low molecular weight saccharide include saccharides having a molecular weight of 1000 or less, such as sucrose, glucose, maltose and the like.

  The inner diameter of the distal end portion 22 on the downstream side in the liquid feeding direction of the inner tube 20 is preferably 0.1 μm to 500 μm, more preferably 1 μm to 100 μm, and still more preferably 5 μm to 30 μm.

  When the inner diameter of the distal end portion 22 of the inner tube 20 is in the above range, the produced droplet follows the law of microfluid dynamics, so that a droplet excellent in monodispersity can be produced more easily.

  In the droplet manufacturing device 100, the inner diameter of the outer tube 10 where the distal end portion 22 of the inner tube 20 is located affects the particle size of the liposome to be produced. For this reason, it is preferable to strictly adjust the central axis of the outer tube 10 and the inner tube 20 and the position of the inner tube 20 inside the outer tube 10. When the inner diameter of the outer tube 10 where the distal end portion 22 of the inner tube 20 is located is 100 to 140 μm, the yield of liposomes tends to increase.

  The constituent material of the outer tube 10 and the inner tube 20 is not particularly limited, and for example, a material such as resin, metal, or glass can be used. Among these, glass is preferable as a constituent material of the outer tube 10 and the inner tube 20. It is easy to produce a tube having a fine inner diameter by heating and stretching the glass tube. For this reason, if the glass tube is cut at a portion having a desired inner diameter, the outer tube 10 and the inner tube 20 having the desired inner diameter can be easily obtained.

  FIG. 6 is a schematic diagram showing how a liposome encapsulating a reagent is manufactured using the above-described droplet manufacturing device 100. As described above, in the droplet manufacturing device 100, the first droplet material is formed in the gap formed between the outer tube 10 and the inner tube 20 in the vicinity of the distal end portion 12 on the downstream side in the liquid feeding direction of the outer tube 10. 10F is in a state where the second droplet material 20F is introduced into the inner tube 20 respectively. The second droplet material 20F includes a substance to be encapsulated in the liposome.

  Here, when the first droplet material 10F and the second droplet material 20F are fed in the direction indicated by the arrows in FIG. 6, the positional relationship between the concentric outer tube 10 and the inner tube 20 is maintained. The first droplet material 10F and the second droplet material 20F are fed as they are, and a laminar flow is formed in which the first droplet material 10F is an outer layer and the second droplet material 20F is an inner layer. The

  The liquid feeding may be performed by, for example, a syringe pump, and may be performed by, for example, the centrifugal force of the centrifuge, but the centrifuge is preferable from the viewpoint that the number of parallel processes is larger than that of the syringe pump.

  When the liquid feeding of the first droplet material 10F and the second droplet material 20F further proceeds, the flow of the second droplet material 20F that is the inner layer becomes discontinuous, and the first droplet material 10F and the second droplet material 20F flow from the front end (downstream side) of the flow. A droplet D1 of the second droplet material 20F is formed.

  The flow of the second droplet material 20F becomes discontinuous because the flow of the first droplet material 10F and the second droplet material 20F becomes unstable due to Rayleigh plateau instability, This is considered to be due to turbulent flow depending on the magnitude of the driving force.

  The droplet D1 has a monolayer of lipid. The droplet D1 further travels straight in the liquid feeding direction, contacts the lipid single membrane M1 formed at the interface between the oil phase O1 and the aqueous phase W1, and reaches the aqueous phase W1 while being surrounded by the lipid single membrane M1. At this time, the droplet D1 has a lipid bilayer membrane. In this way, a reagent-encapsulating liposome D2 having a lipid bilayer similar to that of cells can be obtained.

  Here, by making the second droplet material 20F contain biomolecules such as protein, DNA, and RNA, artificial cells that are functionally similar to cells can be produced.

  Moreover, the density | concentration of the target substance in the inside of the liposome D1 can be controlled by prescribing | regulating the density | concentration of the target substance added to the 2nd droplet material 20F. Various metabolic reactions that normally occur inside and outside the cell can also occur inside the liposome D2.

  Further, by making the lipid contained in the first droplet material 10F and the lipid contained in the oil phase O1 different types, the outer layer and the inner layer of the lipid bilayer membrane can be made asymmetric. it can.

  In the method for producing droplets of the present embodiment, high-quality reagent-encapsulated liposomes with uniform particle sizes can be produced depending on the magnitude of the driving force (for example, centrifugal force). For example, a large amount of reagent-encapsulated liposomes having a diameter of about 0.1 μm to 500 μm can be produced.

  In addition, the produced reagent-encapsulated liposome is again introduced into the inner tube 20 as the second droplet material 20F and fed to form a lipid membrane on the outside of the lipid membrane of the reagent-encapsulated liposome. It can be multilayered. At this time, lipid membranes having the same composition can be multilayered, or lipid membranes having different compositions can be multilayered. For example, by arbitrarily changing the composition of the first droplet material 10F and the second droplet material 20F, a lipid membrane having a composition different from that of the lipid membrane can be formed outside the lipid membrane of the liposome. it can.

[Method of adjusting particle size of droplet]
In one embodiment, the present invention includes an outer tube that feeds a first droplet material, and an inner tube that is disposed inside the outer tube and feeds a second droplet material. There is a gap between the tube and the inner tube, the liquid feeding direction of the first droplet material and the second droplet material is the same, and the downstream side of the liquid feeding direction of the inner tube The first droplet material is fed to the outer tube of the droplet manufacturing device located at the upstream side in the liquid feeding direction with respect to the tip portion of the outer tube on the downstream side in the liquid feeding direction, Including the step of feeding the second droplet material to the inner tube, performing the feeding by centrifuging the device, and adjusting the centrifugal force applied to the device, A method for adjusting the particle size of a droplet is provided.

  As the droplet manufacturing device used in the method of the present embodiment, the same device as described above can be used. It is preferable that the outer tube and the inner tube of the droplet manufacturing device are fixed by the fixing tool 40 described above.

  As will be described later in the embodiments, the particle size of the droplets to be manufactured can be adjusted by adjusting the centrifugal force applied to the droplet manufacturing device. The droplet may be a liposome.

  The magnitude of the centrifugal force applied to the droplet production device may be any value as long as liposomes can be produced, but when droplets are formed due to Rayleigh-plateau instability, it is the same as the inner diameter of the tip of the inner tube Droplets with a particle size are obtained. In addition, if the centrifugal force applied to the droplet manufacturing device exceeds the centrifugal force at which droplets are formed due to Rayleigh plateau instability, droplets with a particle size different from the inner diameter of the tip of the inner tube are obtained. It is done. Thus, the particle size of the droplets can be adjusted by controlling the centrifugal force.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Experimental Example 1]
A water-soluble polymer was added to the second droplet material (droplet material introduced into the inner tube) to prepare liposomes encapsulating magnetic beads. Rhodamine dextran was used as the water-soluble polymer.

(Droplet production device)
In this experimental example, a droplet having a structure similar to that of the droplet manufacturing device 100 using the fixture 40 having a structure similar to that shown in FIGS. 1, 2 (a) and 2 (b). A production device was assembled and used.

(Production of outer tube and inner tube)
Using a puller (manufacturer: Narishige, model number: PC-10), a commercially available glass tube (model “G-1”, manufactured by Narishige; model “1B200-6”, manufactured by World Precision Instruments, Inc.) is stretched out. Tubes and inner tubes were made. The inner diameter of the outer tube tip was 80 μm. Moreover, the inner diameter of the inner tube tip was 10 μm.

(Introduction of droplet material)
15 μL of mineral oil containing 5 mM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was enclosed in the outer tube 10. Further, the tip of the inner tube 10 is immersed in an aqueous solution (test group) containing 1 mg / mL of magnetic beads, rhodamine dextran 5 (w / v)% and sucrose 1M, and 0. 1 μL of aqueous solution was drawn up. “W / v” is the ratio of mass to volume, and “(w / v)%” is the percentage of the ratio of mass to volume. Here, “w” is the mass of rhodamine dextran, and “v” is the volume of the 1M sucrose aqueous solution.

  As a control, an inner tube 10 in which 0.1 μL of an aqueous solution containing no rhodamine dextran (an aqueous solution containing 1 mg / mL magnetic beads and 1 M sucrose) was prepared.

(Fixing inner and outer pipes)
The outer tube 10 and the inner tube 20 filled with the droplet material were sequentially fitted to the fixture 40 described above to assemble a droplet manufacturing device. Further, in order to precisely align the central axes of the outer tube 10 and the inner tube 20, the distance between the distal end 12 of the outer tube 10 and the distal end 22 of the inner tube 20 under the microscope in a state where the fixture 40 is further fixed to the holder. That is, the distance between the outer tube holding member 11 and the inner tube holding member 21 was adjusted.

  After moving the inner tube holding member 21 so that the inner diameter of the outer tube 10 where the inner tube 20 distal end 22 is located becomes 115 μm, the fixing screw 32 and the fixing screw 33 were tightened and fixed firmly.

(Preparation for droplet recovery)
30 μL of 1 M glucose (aqueous phase) was poured into a commercially available 1.5 mL tube (model “MP-150HC”, manufactured by Nikyo Technos Co., Ltd.), and 100 μL of DOPC 5 mM-containing mineral oil was added from above (oil phase). After leaving still for a while so that lipids may be arranged in a single layer, the fixture 40 to which the outer tube 10 and the inner tube 20 were fixed was housed inside the tube. The amount of mineral oil was finely adjusted so that the distance between the tip 12 of the outer tube 10 and the oil phase liquid level was 6 mm.

(Preparation of liposome encapsulating magnetic beads)
Set the above tube in a tabletop small centrifuge (model “ATT-101”, manufactured by Hitec Co., Ltd.), centrifuge for 10 seconds with the maximum output centrifugal force, external liquid (mineral oil containing 5 mM DOPC) and internal The liquid (the test group or the control aqueous solution described above) was simultaneously fed to produce droplets containing magnetic beads due to Rayleigh plateau instability. The produced droplets enclosing the magnetic beads passed through a lipid membrane between mineral oil and water stored at the bottom of the tube, became lipid bilayer liposomes, and were released into the water at the bottom of the tube.

(Confirmation of liposome encapsulating magnetic beads)
The produced liposomes were collected and observed with a fluorescence microscope. FIGS. 7A to 7D are micrographs of liposomes. FIG. 7 (a) is a photomicrograph in the bright field of control liposomes. FIG. 7B is a fluorescence micrograph of the fluorescence of the magnetic beads observed in the same field of view as FIG. As a result, as shown in FIG. 7 (a), the control liposomes had less formation than the test group described later. In addition, the presence of liposomes was confirmed in the circled region in FIG. 7A, but the fluorescence of the magnetic beads was observed in the circled region in FIG. 7B corresponding to this region. Was not. From this result, it is clear that when liposomes are prepared using the second droplet material that does not contain a water-soluble polymer, the amount of liposomes formed is small and the efficiency of encapsulating the target substance (magnetic beads) in the liposomes is also low. It became.

  Moreover, FIG.7 (c) is a microscope picture in the bright field of the liposome of a test group. FIG. 7 (d) is a fluorescence micrograph of the fluorescence of the magnetic beads observed in the same field of view as FIG. 7 (c). As a result, as shown in FIG. 7C, the amount of the liposomes in the test group was larger than that of the control, and about 100 liposomes were confirmed in one field of view. Moreover, as shown in FIG.7 (d), the position of the fluorescence in the fluorescence microscope photograph which observed the fluorescence of the bright field liposome and the magnetic substance bead corresponded. Furthermore, the fluorescence moved when a neodymium magnet was brought closer under the microscope. The above results indicate that by adding a water-soluble polymer to the second droplet material, a large amount of liposomes in which magnetic beads are encapsulated with high efficiency could be produced.

[Experiment 2]
Liposome was produced by assembling a droplet production device described in Patent Document 1 in which the inner tube and the outer tube were fixed using a conventional fixture, and compared with the droplet production device of Experimental Example 1.

  As a conventional fixture, the same fixture as the fixture 40 'shown in FIG. 3 was used. The fixture 40 ′ includes the outer tube holding member 11 and the inner tube holding member 21 similarly to the fixture 40 of Experimental Example 1, and is different from the connecting member 30 in the fixture 40 of Experimental Example 1, The connecting member 30 ′, which is a screw, was provided. For this reason, the distance between the outer tube holding member 11 and the inner tube holding member 21 can be adjusted only by a multiple of the distance between the threads of the connecting member 30 ′. Further, in the fixture 40 ′, since the outer tube 10 and the inner tube 20 need to be disposed after adjusting the distance between the outer tube holding member 11 and the inner tube holding member 21, the outer tube is observed while observing under a microscope. The distance between the holding member 11 and the inner tube holding member 21 could not be adjusted. Further, in the fixture 40 ′, the outer tube holding member 11 and the inner tube holding member 21 cannot be fixed, and the centers of the inner tube 20 and the outer tube 10 cannot be matched.

(Preparation of liposome)
15 μL of mineral oil containing 5 mM of DOPC was sealed inside the outer tube. The tip of the inner tube was immersed in an aqueous solution containing rhodamine dextran 5 (w / v)% and sucrose 1M, and 0.1 μL of the aqueous solution was sucked into the inner tube by capillary action.

  Subsequently, a droplet production device was assembled using a conventional fixture or a fixture similar to Experimental Example 1, respectively. The inner diameter of the outer tube where the inner tube tip is located was about 110 μm in any droplet production device. Subsequently, liposomes were produced in the same manner as in Experimental Example 1 using each droplet production device.

(Confirmation of the number of liposomes)
The produced liposomes were collected and observed with a fluorescence microscope. FIG. 8 (a) is a fluorescence micrograph of liposomes produced with a droplet production device assembled using a conventional fixture. FIG. 8B is a fluorescence micrograph of liposomes produced with a droplet production device assembled using the same fixture as in Experimental Example 1. In both FIGS. 8 (a) and (b), the fluorescence of rhodamine was observed.

  As a result, an average of 70 liposomes were confirmed per 10 μL of the aqueous phase in the droplet production device assembled using the conventional fixture. On the other hand, in the droplet production device assembled using the same fixture as in Experimental Example 1, an average of 500 liposomes was confirmed per 10 μL of the aqueous phase. Thus, by using the same fixture as in Experimental Example 1, it was possible to increase the amount of liposome formation by about 7 times.

[Experiment 3]
Using a droplet production device assembled using the same fixture as in Experimental Example 1, the centrifugal conditions were changed to prepare liposomes.

(Preparation of liposome)
15 μL of mineral oil containing 5 mM of DOPC was sealed inside the outer tube. The tip of the inner tube was immersed in an aqueous solution containing rhodamine dextran 5 (w / v)% and sucrose 1M, and 0.1 μL of the aqueous solution was sucked into the inner tube by capillary action.

  Subsequently, a droplet production device was assembled using the same fixture as in Experimental Example 1. The inner diameter of the outer tube where the inner tube tip is located was about 110 μm in any droplet production device. Subsequently, 30 μL of 1 M glucose (aqueous phase) was poured into a commercially available 1.5 mL tube (model “MP-150HC”, manufactured by Nikyo Technos Co., Ltd.), and 100 μL of DOPC5 mM-containing mineral oil was added from above (oil phase). . After standing for a while so that the lipids were lined up in a layer, the assembled droplet production device was housed inside the tube. The amount of mineral oil was finely adjusted so that the distance between the tip of the outer tube and the oil phase liquid level was 6 mm.

  Subsequently, the above tube is set in a high-speed centrifuge (manufactured by Kubota), and the centrifuge conditions are controlled to 500 × g (g means gravitational acceleration) or 1000 × g, followed by centrifugation for 10 seconds. Was made.

(Confirmation of the number of liposomes)
The produced liposomes were collected and observed with a fluorescence microscope, and the particle size and number of the liposomes were measured. FIG. 9A is a graph showing the measurement results of the particle size and abundance of liposomes prepared under 500 × g centrifugation conditions. The same experiment was performed twice. FIG.9 (b) is a graph which shows the measurement result of the particle size and abundance rate of the liposome produced on 1000 * g centrifugation conditions. The same experiment was performed twice.

  As a result, liposomes having a particle size similar to the inner tube size (the inner diameter of the inner tube tip of 10 μm) and smaller particles at 500 × g, which is smaller than the rate at which laminar flow based on Rayleigh plateau instability occurs, are obtained. Diameter-sized liposomes were produced with approximately the same abundance.

  On the other hand, at 1000 × g, which is larger than the rate at which laminar flow based on Rayleigh plateau instability occurs, the abundance of liposomes having a particle size smaller than the inner tube size (the inner diameter of the inner tube tip is 10 μm) was higher.

  This result shows that the particle size of the liposome can be controlled by controlling the centrifugal force even in the droplet production device using the inner tube of the same size. That is, by controlling the centrifugal force, a liposome having a small particle size can be produced from an inner tube having a large inner diameter.

  The configurations and combinations thereof in the embodiments described above are examples, and additions, omissions, substitutions, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  According to the present invention, an improved liposome forming technique can be provided. The present invention can be widely used in chemistry-related fields, biological-related fields, etc., and can also be used for biochemical synthesis, gene analysis, and the like. In addition, as shown in the examples, the present invention can be used for protein synthesis in droplets and production of artificial cell membrane vesicles (liposomes), and thus is particularly suitable in the fields related to biology and medicine. Is available. In particular, liposomes having a structure closer to artificial cells, such as microdomains, asymmetry of the inner and outer layers of the membrane, and the introduction of membrane proteins, can be produced.

  DESCRIPTION OF SYMBOLS 10 ... Outer tube, 11 ... Outer tube holding member, 11 ', 21' ... Support body, 11a, 11'a, 21a, 21'a ... Hole, 12, 22 ... Tip part, 20 ... Inner tube, 21 ... Inside Tube holding member, 30, 30 '... coupling member, 31 ... pin, 32, 33 ... fixing screw, 40, 40' ... fixture, 100 ... droplet production device, 10F ... first droplet material, 20F ... Second droplet material, D1 ... droplet, D2 ... reagent-encapsulating liposome, O1 ... oil phase, W1 ... aqueous phase, M1 ... lipid membrane.

Claims (5)

An outer tube that feeds the first droplet material; and an inner tube that is disposed inside the outer tube and feeds the second droplet material, and is disposed between the outer tube and the inner tube. Has a gap, the liquid feeding direction of the first droplet material and the second liquid droplet material is the same, and the tip of the inner tube on the downstream side in the liquid feeding direction is the A fixture for fixing the inner tube to the inside of the outer tube of the droplet manufacturing device located on the upstream side in the liquid feeding direction with respect to the tip portion on the downstream side in the liquid feeding direction,
An outer tube holding member for holding the outer tube;
An inner tube holding member for holding the inner tube;
A connecting member that connects the outer tube holding member and the inner tube holding member,
The connecting member holds the outer tube in the state where the outer tube is held by the outer tube holding member, the inner tube is held by the inner tube holding member, and the inner tube is disposed inside the outer tube. The distance between the member and the inner tube holding member in the liquid feeding direction is connected to be adjustable.
Fixture.   The fixture according to claim 1, which is made of metal. An outer tube that feeds the first droplet material; and an inner tube that is disposed inside the outer tube and feeds the second droplet material, and is disposed between the outer tube and the inner tube. Has a gap, the liquid feeding direction of the first droplet material and the second liquid droplet material is the same, and the tip of the inner tube on the downstream side in the liquid feeding direction is the The first droplet material is fed to the outer tube and the second liquid is fed to the inner tube of the droplet manufacturing device located on the upstream side in the liquid feeding direction from the tip on the downstream side in the liquid feeding direction. Including a step of feeding a drop material,
The droplet is a liposome, the first droplet material includes a lipid constituting a membrane of the liposome, and the second droplet material includes a hydrophilic polymer.
Method for producing droplets.   The method for producing droplets according to claim 3, wherein the hydrophilic polymer is a polysaccharide or a polyether. An outer tube that feeds the first droplet material; and an inner tube that is disposed inside the outer tube and feeds the second droplet material, and is disposed between the outer tube and the inner tube. Has a gap, the liquid feeding direction of the first droplet material and the second liquid droplet material is the same, and the tip of the inner tube on the downstream side in the liquid feeding direction is the The first droplet material is fed to the outer tube and the second liquid is fed to the inner tube of the droplet manufacturing device located on the upstream side in the liquid feeding direction from the tip on the downstream side in the liquid feeding direction. Including a step of feeding a drop material,
The liquid feeding is performed by centrifuging the device, and by adjusting the centrifugal force applied to the device, the particle size of the droplets to be manufactured is adjusted.
A method of adjusting the droplet size.