JP2016198760A - Method of producing bubble - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing bubbles that enables bubbles (micro-bubbles or nano-bubbles) having a uniform size to be stably produced.SOLUTION: A method of producing bubbles has steps for: injecting a solution containing an object substance into a prescribed height of a container; and vibrating the container at the speed of rotation of 5,000 rpm or higher so that the solution collides repeatedly with the inner surface of the container in the state where the inside of the container is set at a pressure higher than 1.0 atm.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a method for producing microbubbles or nanobubbles, and a bubble production container used in the method for producing microbubbles or nanobubbles. In particular, the present invention relates to a method for producing microbubbles or nanobubbles used for ultrasonic diagnosis and ultrasonic therapy, and a bubble production container used for the method for producing microbubbles or nanobubbles.

  In recent years, utilization of micron-sized (several hundred microns or less) or nano-sized (several hundred nanometers or less) bubbles has been studied in various fields such as medical care, food, seafood culture, and wastewater treatment. In particular, in the medical field, a method for ultrasonic diagnosis of the chest and abdomen using microbubbles as an ultrasound contrast agent is known.

  This ultrasonic diagnostic method is a method in which an ultrasonic contrast agent is injected into the body from a vein or the like, ultrasonic irradiation is performed on the diagnostic region, and a reflected wave (reflected echo) from the ultrasonic contrast agent is imaged and diagnosed. is there. And as an ultrasonic contrast agent, the micro bubble (micro bubble) which consists of the outline comprised from protein, lipid, etc., and the gas enclosed in the outline is widely used.

  In recent years, an ultrasonic treatment method using the microbubbles has been studied (for example, Patent Document 1). More specifically, microbubbles encapsulating genes and drugs (drugs) are injected into the body and carried through the blood vessels to the affected area. When the microbubble reaches the vicinity of the affected part, the microbubble is ruptured by irradiating the microbubble with an ultrasonic wave. By doing so, the drug enclosed in the microbubbles can be intensively administered to the affected area.

  As a method for producing such microbubbles, a supersaturated bubble generation method and a gas-liquid two-phase flow swirl method are known. The supersaturated bubble generation method is a method in which a microbubble is generated in a liquid mixture by dissolving a gas under high pressure in a liquid mixture containing a constituent material of microbubbles and physiological saline and then reducing the pressure. In the gas-liquid two-phase flow swirl method, the above mixed liquid is stirred at a high speed to generate a vortex of the mixed liquid. After sufficiently entraining the gas in the vortex, mixing is performed by stopping the vortex. In this method, microbubbles are generated in the liquid.

  In the microbubble manufacturing method, it is necessary to prepare a mixed liquid of 1 to 10 L at least in order to generate microbubbles. In addition, it is difficult to stably generate microbubbles with a small amount of liquid mixture (for example, several ml of liquid mixture). In addition, there is a problem that the size of the generated microbubbles varies.

JP 2002-209896 A

  The present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide a bubble manufacturing method capable of stably manufacturing bubbles of uniform size (microbubbles or nanobubbles). . Another object is to provide a bubble manufacturing container used in a method for manufacturing a bubble of uniform size.

Such an object is achieved by the present inventions (1) to (18) below.
(1) Injecting a solution containing the target substance to a predetermined height of the container;
And vibrating the container at a rotational speed of 5000 rpm or more so that the solution repeatedly collides with the inner surface of the container in a state where the pressure in the container is larger than 1.0 atm. The bubble manufacturing method.

  (2) The bubble manufacturing method according to (1), wherein the step of vibrating the container includes a step of generating a shock wave by causing the solution to collide with an inner surface of the container.

  (3) The bubble production method according to (2), wherein the pressure of the shock wave is 40 kPa to 1 GPa.

  (4) The bubble manufacturing method according to any one of (1) to (3), wherein the step of vibrating the container is performed so as to vibrate the container by a reciprocating motion and / or a rotating motion.

  (5) The step of vibrating the container is performed such that the container is vibrated by reciprocating and / or rotating movement of the container in a horizontal direction and / or a vertical direction. The method for producing a bubble according to any one of the above.

  (6) The step of vibrating the container is performed so as to vibrate the container so that an instantaneous relative speed between the container and the solution is 40 km / h or more when the solution collides with the inner surface of the container. The method for producing a bubble according to any one of (1) to (5) above.

  (7) The bubble manufacturing method according to any one of (1) to (6), wherein the step of vibrating the container is performed in a state where the pressure is set to 1.5 to 10 atm.

  (8) The predetermined height is the height of the liquid surface of the solution in the container, where the height of the container is X (mm) in a state where the container into which the solution has been injected is left still horizontally. The method for producing bubbles according to any one of the above (1) to (7), wherein the relationship of 0.2 ≦ Y / X ≦ 0.7 is satisfied, where Y is (mm).

  (9) Any of the above (1) to (8), further comprising the step of revibrating the container at a rotational speed of 5000 rpm or higher after changing the pressure in the container after the step of vibrating the container The manufacturing method of the bubble as described in 2.

  (10) The bubble manufacturing method according to (9), wherein the step of vibrating the container again is performed such that the pressure is higher by 1 to 10 atm than the pressure in the step of vibrating the container.

  (11) The bubble manufacturing method according to (9) or (10), wherein the step of re-vibrating the container is performed at a rotational speed different from the rotational speed in the step of vibrating the container.

  (12) The step of injecting the solution into the container and the step of vibrating the container are performed in any one of the above (1) to (11) performed so as to maintain a constant temperature of the solution. Bubble manufacturing method.

  (13) The bubble manufacturing method according to any one of (1) to (12), wherein the target substance includes an outer material that forms an outer shape of the bubble.

  (14) The outer shell material is an amphiphilic material containing at least one of proteins, polycationic lipids, phospholipids, higher fatty acids, saccharides, sterols, surfactants, natural and synthetic polymers. The method for producing a bubble according to (13).

  (15) The outer shell is composed of a micelle composed of a monolayer of molecules of the amphiphilic material, or a liposome composed of a bilayer of molecules of the amphiphilic material. Bubble manufacturing method.

  (16) The bubble production method according to any one of (1) to (15), wherein the target substance includes a drug.

  (17) The bubble production method according to any one of (1) to (16), wherein an average particle diameter of the bubbles is 10 nm to 1000 μm.

  (18) A bubble manufacturing container used in the bubble manufacturing method according to any one of (1) to (17).

  According to the present invention, it is possible to stably generate a large amount of uniform-sized bubbles in a solution simply by vibrating the container while the inside of the container is pressurized. As a result, a container containing a large amount of uniform-sized bubbles can be provided.

It is a perspective view which shows the state which cut | disconnected some bubbles manufactured by the manufacturing method of the bubble of this invention. FIG. 1 (a) shows a state where a part of a bubble in which gas is sealed in the outer shell is cut, and FIGS. 1 (b) and 1 (c) show that gas and drug are sealed in the outer shell. A state in which a part of the bubble is cut is shown. It is a flowchart for demonstrating 1st Embodiment of the manufacturing method of the bubble of this invention. 3A to 3D are cross-sectional views for explaining the first embodiment of the bubble manufacturing method of the present invention. FIG. 4 is a partially enlarged view for explaining a state in which the mixed liquid and the inner surface (upper surface) of the container collide violently in the step of vibrating the container shown in FIG. It is a flowchart for demonstrating 3rd Embodiment of the manufacturing method of the bubble of this invention. It is a fragmentary sectional view which shows the cover vicinity of the manufacturing container used for 4th Embodiment of the manufacturing method of the bubble of this invention. FIGS. 7A to 7F are cross-sectional views schematically showing another configuration of the container used in the fifth embodiment of the bubble manufacturing method of the present invention. FIGS. 8A to 8C are perspective views for explaining a sixth embodiment of the bubble manufacturing method of the present invention. It is a figure for demonstrating the structure (The handle | steering-wheel is abbreviate | omitted) vicinity of the rubber stopper of the mini-art valve shown in FIG. 9A is a top view of the vicinity of the rubber plug of the mini-nert valve, and FIG. 9B is a cross-sectional view taken along the line XX of FIG. 9A. It is sectional drawing of the bubble containing container shown in FIG.8 (c). It is a perspective view for demonstrating the manufacturing container used for 7th Embodiment of the manufacturing method of the bubble of this invention. It is sectional drawing for demonstrating the manufacturing container used for 8th Embodiment of the manufacturing method of the bubble of this invention. It is sectional drawing for demonstrating the manufacturing container used for 9th Embodiment of the manufacturing method of the bubble of this invention. FIG. 13A shows the manufacturing container in an exploded state, and FIG. 13B shows the manufacturing container in an assembled state. It is sectional drawing for demonstrating the manufacturing container used for 10th Embodiment of the manufacturing method of the bubble of this invention. FIG. 14A shows the manufacturing container in an exploded state, and FIG. 14B shows the manufacturing container in an assembled state. It is a figure for demonstrating the position of the opening part formed in the lid | cover of a manufacturing container shown in FIG.14 (b). FIG. 15 (a) is a view for explaining a state before the injection needle of the syringe is punctured into the rubber stopper, and FIG. It is a figure for demonstrating the state fastened to the part. FIG. 16A is a graph showing the particle size distribution of the bubbles when the bubbles are produced at a rotational speed of 5000 rpm and 6500 rpm. FIG.16 (b) is the elements on larger scale whose horizontal axis is the range of 0-700 nm in the graph shown to Fig.16 (a). FIG. 17A is a graph showing the relationship between the number of rotations of the closed vial and the average particle diameter of the bubbles. FIG. 17B is a graph showing the relationship between the rotational speed of the closed vial and the bubble content. FIG. 18 (a) is a graph showing the relationship between the volume of gas sealed in a closed vial and the average particle diameter of bubbles. FIG. 18B is a graph showing the relationship between the volume of gas sealed in the closed vial and the bubble content. It is a fluorescence-microscope image of the cerebral vascular pericyte cell culture medium cultured at 37 degreeC for 48 hours. FIG. 19 (a), irradiation intensity: 0.6 W / cm a ultrasonic irradiation image of the sample at ^2, FIG. 19 (b), irradiation intensity: at 0.8 W / cm ² of ultrasonic irradiation samples It is an image. It is a fluorescence-microscope image of the cerebral vascular pericyte cell culture medium cultured at 37 degreeC for 48 hours. 20 (a) is irradiation intensity: 0.9 W / cm ² in a ultrasonic irradiation image of the sample, FIG. 20 (b), irradiation intensity: at 1.0 W / cm ² of ultrasonic irradiation samples It is an image.

  Hereinafter, a bubble manufacturing method and a bubble manufacturing container of the present invention will be described based on preferred embodiments shown in the accompanying drawings.

  First, prior to the description of the bubble manufacturing method and bubble manufacturing container of the present invention, the bubble manufactured by the bubble manufacturing method of the present invention will be described.

  FIG. 1 is a perspective view showing a state where a part of a bubble manufactured by the bubble manufacturing method of the present invention is cut. FIG. 1 (a) shows a state in which a part of a bubble in which gas is sealed in the outer shell is cut, and FIGS. 1 (b) and 1 (c) show that gas and drug are in the outer shell. The state which cut some encapsulated bubbles is shown.

First, the bubble 1 shown in FIG.
The bubble 1 (bubble) shown to Fig.1 (a) can be manufactured by the 1st, 3-10 embodiment of the manufacturing method of the bubble of this invention mentioned later. Such a bubble 1 has an outer shell 2 (bubble film) constituting the shell of the bubble 1 and a gas 3 sealed in the outer shell 2. Such a bubble 1 can be used in various fields such as medical care, food, seafood culture, and wastewater treatment. In the present embodiment, a case where the bubble 1 is used as an ultrasonic contrast agent in ultrasonic diagnosis will be described. Hereinafter, each component which comprises the bubble 1 is demonstrated.

The outer shell 2 has a function of holding the gas 3 sealed inside thereof in the bubble 1.
Such a shell 2 is composed of an amphiphilic material (outer material) having both hydrophobic and hydrophilic properties (substituents) in one molecule. The amphiphilic material is not particularly limited. For example, proteins such as albumin, polycationic lipids, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phospholipids such as phosphatidylethanolamine, palmitic acid, stearic acid Higher fatty acids such as saccharides, saccharides such as galactose, cholesterol, sterols such as sitosterol, surfactants, natural or synthetic polymers, fluorescent dyes, antibodies, labeled metals, etc. Two or more kinds can be used in combination.

  Although not shown in FIG. 1, the amphiphilic material constituting the outer shell 2 is arranged in a spherical shape in the aqueous medium so that the hydrophobic group is on the inner side and the hydrophilic group is on the outer side. Due to this property, the outer shell 2 becomes a micelle composed of a single layer of amphiphilic material molecules or a liposome composed of a double layer of amphiphilic material molecules.

  The gas 3 has a function of stabilizing the outer shell 2 of the bubble 1. Such a gas 3 is a gaseous substance at a temperature (about 20 ° C.) when the bubble 1 is manufactured. Further, such a bubble 1 is a gaseous substance even when the bubble 1 is injected into the body, that is, at a temperature inside the body (about 37 ° C.).

  Such gas 3 is not particularly limited, but for example, air, nitrogen, oxygen, carbon dioxide, hydrogen, helium, argon, xenon, krypton inert gas, sulfur hexafluoride, disulfur decafluoride , Sulfur fluoride such as trifluoromethyl sulfur pentafluoride, methane, ethane, propane, butane, pentane, cyclopropane, cyclobutane, cyclopentane, ethylene, propylene, propadiene, butene, acetylene, propyne, perfluoropropane, perfluoropropane Low molecular weight hydrocarbons such as fluorobutane and perfluoropentane, or halides thereof, ethers such as dimethyl ether, ketones, esters, etc. may be mentioned, and one or more of these may be combined. Can be used. Among these substances, sulfur hexafluoride, perfluoropropane, perfluorobutane, and perfluoropentane are particularly preferable. The bubble 1 encapsulating these gases has high stability in the body and is more reliably transported through the blood vessel to the affected area or the diagnosis site.

  The particle diameter of the bubble 1 composed of such components is changed by changing the conditions of each step of the bubble production method of the present invention. That is, the produced bubble 1 has a micron size (several hundred microns or less) or a nanosize (several hundred nanometers or less).

  Specifically, the average particle diameter (also referred to as the outer average particle diameter) of the bubble 1 is not particularly limited, but is preferably about 10 nm to 1000 μm, more preferably about 10 nm to 10 μm, and more preferably 50 to 2000 nm. More preferably, it is about. If the average particle size of bubble 1 is within the above range, when bubble 1 is injected into the body by intravenous injection, the particle size of bubble 1 is sufficiently small, so that bubble 1 moves smoothly in the blood vessel due to blood flow. can do. In addition, the bubble having such a particle size has high stability in the blood vessel, and is surely carried to the target site without disappearing while moving in the blood vessel.

  In general, a bubble containing gas has a property of efficiently reflecting ultrasonic waves at the interface between the outer shell and the gas. Therefore, if the particle size of the bubble 1 is within the above range, the area of the interface between the outer shell 2 and the gas 3 is sufficiently large, so that the bubble 1 is effectively used as an ultrasound contrast agent.

  The bubble 1 having the above-described configuration can also be used in fields other than the medical field such as food, seafood culture, and wastewater treatment. In particular, if the average particle size of the bubble 1 is within the above range, the stability of the bubble 1 can be sufficiently increased. Since the highly stable bubble 1 is easy to handle, it can be applied to various fields.

Next, the bubble 1 shown in FIG.1 (b) and FIG.1 (c) is demonstrated.
The bubble 1 shown in FIG.1 (b) and FIG.1 (c) can be manufactured by 2nd, 3-10 embodiment of the manufacturing method of the bubble of this invention mentioned later. Such a bubble 1 has an outer shell 2 constituting the shell of the bubble 1, and a gas 3 and a drug 4 enclosed in the outer shell 2. Such a bubble 1 is used for ultrasonic therapy and ultrasonic diagnosis. 1B shows the bubble 1 in which the drug 4 is sealed in the outer shell 2 in a gas state or a solid state, and in FIG. 1C, the drug 4 is in the outer shell 2 in the liquid state. An encapsulated bubble 1 is shown.

  The outer shell 2 has a function of holding the gas 3 and the drug 4 sealed inside thereof in the bubble 1 and has a function of protecting the drug 4 until the bubble 1 is carried to the affected part. As the material constituting the outer shell 2, the same material as that of the outer shell 2 of the bubble 1 shown in FIG.

  The gas 3 has a function of stabilizing the outer shell 2 of the bubble 1. As the gas 3, the same material as the gas 3 of the bubble 1 shown in FIG.

  In the bubble 1 shown in FIGS. 1B and 1C, the drug 4 is an effective component for treating various diseases such as prostate cancer, uterine fibroids, myocardial infarction, cerebral infarction and the like. Such a drug 4 is conveyed to the affected part in a state of being included in the bubble 1 and is administered to the affected part by rupturing the bubble 1 near the affected part by ultrasonic irradiation. The drug 4 is enclosed in the outer shell 2 together with the gas 3, or is contained or adsorbed in the outer shell 2 itself.

  Such a drug 4 is not particularly limited as long as it is effective for treating diseases, but includes a gene, a drug, and the like. Specifically, peptides, antibodies, oligosaccharides, polysaccharides, genes, oligonucleotides, antisense oligonucleotides, siRNA, ribozymes, triple helix molecules, viral vectors, plasmids, low molecular organic compounds, anticancer agents, metals, etc. One or more of these can be used in combination.

  When bubble 1 contains drug 4, the content of drug 4 is not particularly limited. However, the volume ratio of the drug 4 to the gas 3 is preferably about 1:99 to 90:10, more preferably about 10:90 to 70:30, and about 40:60 to 60:40. More preferably. If the volume ratio of the drug 4 and the gas 3 is within the above range, the stability of the bubble 1 is improved, and the bubble 1 can be more reliably transported to the vicinity of the affected part. In addition, when the bubble 1 is ruptured near the affected area, a sufficient amount of drug can be administered to the affected area. Therefore, the affected part can be treated more efficiently.

  The type and content of the drug 4 to be included in the bubble 1 are appropriately determined according to the type of disease, the size of the affected part, and the like.

  The particle size of the bubble 1 composed of such components is changed by changing the conditions of each step of the bubble production method of the present invention, as in the bubble 1 shown in FIG.

  In particular, if the average particle size of the bubble 1 is 200 to 300 nm, it can be suitably used for cancer treatment. Specifically, in an affected part where cancer cells are present, there is a new blood vessel having a diameter smaller than that of a normal blood vessel that extends from the surrounding blood vessel to the cancer cell. If the average particle diameter of the bubble 1 is within the above range, the bubble 1 can be transported also into a new blood vessel having a small diameter. Therefore, the bubble 1 can be directly conveyed to the cancer cell through the new blood vessel. If the average particle diameter of the bubbles 1 is within the above range, some of the bubbles 1 can be directly transported to cancer cells through the blood vessel wall. In addition, if the average particle size of the bubble 1 is 600 to 900 nm, the bubble 1 can move smoothly in the blood vessels of the brain, and the position of the bubble 1 can be reliably determined when an ultrasonic image is taken. Can be identified. Therefore, it can be suitably used for brain treatment (for example, intravascular treatment).

  The average particle diameter of the bubble 1 shown in FIGS. 1A to 1C is observed by, for example, a laser diffraction / scattering method, a nanoparticle tracking analysis method, an electric resistance method, an AFM (Atomic Force Microscope), or a laser microscope. It can measure by. As an apparatus for measuring AFM, for example, a resonance type particle measurement system (trade name: Archimedes) manufactured by Malvern may be used.

  The bubble 1 as described above can be manufactured by the bubble manufacturing method of the present invention described below. Hereinafter, the bubble production method of the present invention will be described in detail.

<First Embodiment>
1. Next, a first embodiment of the bubble manufacturing method of the present invention will be described. The bubble 1 shown in FIG. 1A can be manufactured by the bubble manufacturing method of the present embodiment.

  FIG. 2 is a flowchart for explaining a first embodiment of the bubble manufacturing method of the present invention, and FIGS. 3A to 3D illustrate the first embodiment of the bubble manufacturing method of the present invention. FIG. 4 is a partially enlarged view for explaining a state in which the mixed solution and the inner surface (upper surface) of the container collide violently in the step of vibrating the container shown in FIG. is there.

  In the following description, the upper side in FIGS. 3A to 3D and 4 is referred to as “upper”, and the lower side in FIGS. 3A to 3D and 4 is referred to as “lower”. say.

  As shown in FIG. 2, the bubble manufacturing method according to the present embodiment includes five steps of step (S1) to step (S5). In the step (S1), a solution (mixed liquid) containing a target substance (outer material constituting the outer shell 2 described above) and a bubble manufacturing container (hereinafter simply referred to as “manufacturing container”) for injecting the mixed liquid It is a process to prepare. Step (S2) is a step of injecting this mixed solution to a predetermined height of the production container. Step (S3) is a step of sealing the manufacturing container in a state where the manufacturing container is filled with gas and the inside of the manufacturing container is pressurized. Step (S4) is a step of vibrating the production container at a predetermined rotational speed so that the mixed solution repeatedly collides with the inner surface of the container. Step (S5) is a step of standing the production container. Hereinafter, these steps will be sequentially described.

[S1] Preparatory Step First, a material (outer material) constituting the outer shell 2 of the bubble 1 and an aqueous medium are placed in a mixed solution preparation container (hereinafter simply referred to as “preparation container”), and the outer material is dissolved in the aqueous medium. To prepare a mixed solution 10. That is, after adding a predetermined amount of the outer material and the aqueous medium in the preparation container, the outer material is stirred to dissolve the outer material in the aqueous medium. The order in which the outer shell material and the aqueous medium are put into the preparation container is not particularly limited. As a method for dissolving the outer shell material in the aqueous medium, for example, stirring with a stirring bar, ultrasonic treatment, or the like can be used.
As the outer material, the above-mentioned amphiphilic material is used.

  The content of the outer material in the mixed liquid 10 is not particularly limited as long as the bubble 1 is formed in the mixed liquid 10 in the step (S4). The preferable content of the outer material varies depending on the combination of the outer material and the aqueous medium, but the outer material is preferably contained in the liquid mixture 10 at a concentration equal to or higher than the critical micelle concentration (CMC). Specifically, the content of the outer material contained in the mixed liquid 10 is preferably 0.01 to 50 wt%, and more preferably 0.1 to 20 wt%.

  Thereby, since the density | concentration of the outline material in the liquid mixture 10 becomes more than a critical micelle density | concentration more reliably, the outline 2 (liposome, micelle) can be reliably formed in the liquid mixture 10. Therefore, in the step (S4) described later, the gas 3 is easily taken into the liposome or micelle, and the bubble 1 having a desired particle diameter can be easily generated in the mixed solution 10. Moreover, the stability of the outer shell 2 is increased, and the bubble 1 can be more reliably prevented from unintentionally bursting. Furthermore, the variation in the size of the generated bubble 1 can be reduced. That is, the bubble 1 having a uniform size can be generated.

  The aqueous medium is not particularly limited, and for example, water (distilled water), physiological saline (phosphate buffered saline), a sucrose solution in which sucrose and distilled water are mixed, and the like can be used.

  The content of the aqueous medium contained in the prepared mixed liquid 10 is preferably 50 to 99.99 wt%, and more preferably 80 to 99.0 wt%. Thereby, the outer shell material can be sufficiently dissolved in the aqueous medium, and a more uniform mixed liquid 10 can be obtained.

Next, the manufacturing container 20 (the container for bubble production of this embodiment) is prepared.
The production container 20 includes a container main body 21 that contains the mixed liquid 10 and a lid 22 for sealing the container main body 21.

  Although the container main body 21 is not specifically limited, It is preferable that the external shape as shown to Fig.3 (a) has comprised the cylindrical shape. In the present embodiment, a vial with a capacity of about 0.5 to 20 ml is used as the container body 21. In the bubble manufacturing method of the present invention, even when such a small-sized vial is used as the container body 21, when the container body 21 is sealed with the lid 22, the sealed space in the container body 21 is used. Since an appropriate pressure is applied to the liquid mixture 10, uniform size bubbles can be stably obtained. In particular, in the case of a vial having a capacity of about 0.5 to 1.5 ml, a bubble-containing mixed solution of about 0.3 to 0.6 ml that is required for one ultrasonic diagnosis in one manufacturing container 20 Can be manufactured. In this case, since the bubble-containing mixed solution in one manufacturing container 20 can be used up in ultrasonic diagnosis, waste of the bubble-containing mixed solution to be manufactured can be eliminated.

  Thus, the small capacity vial (capacity: about 0.5 to 20 ml) has a height X of about 35 to 60 mm and an outer diameter R of about 10 to 40 mm.

  As shown in FIGS. 3B to 3D, the lid 22 fixes a disc-shaped rubber stopper (septum) 221 that is in close contact with the bottle mouth of the container body 21 and the rubber stopper 221 to the bottle mouth of the container body 21. And a tightening portion 222 to be tightened.

  The rubber stopper 221 is not particularly limited, and for example, a rubber stopper made of silicon can be used.

The tightening portion 222 is configured to cover the edge of the rubber plug 221. In addition, screw grooves formed so as to be screwable with each other are formed on the inner peripheral surface on the bottle mouth side of the tightening portion 222 and on the outer peripheral surface on the bottle mouth side of the container body 21 (not shown). The rubber plug 221 is fixed in close contact with the bottle mouth of the container body 21 by screwing them together. Further, the container body 21 and the tightening part 222 can be fixed in a state where the rubber stopper 221 is in close contact with the bottle mouth of the container body 21 by caulking the fastening part 222 to the bottle mouth of the container body 21. .
The preparation container and the production container 20 may be different containers or the same container.

  When the preparation container and the production container 20 are different containers, for example, as the preparation container, a container having a relatively large capacity is used, while the production container 20 (container body 21) has a relatively small capacity. Containers can be used. In this case, by preparing a large amount of the liquid mixture 10 having a uniform composition in the preparation container and subdividing the liquid mixture 10 into a plurality of production containers 20, the size of bubbles generated in each production container 20 ( Particle size) and quantity can be made uniform.

Further, when the preparation container and the production container 20 are the same container, the step (S2) can be omitted. Therefore, there is an advantage in that the process can be simplified.
In the present embodiment, a container different from the manufacturing container 20 is used as the preparation container.

[S2] Step of injecting the mixed liquid into the production container The prepared mixed liquid 10 is injected to a predetermined height of the container body 21 (production container 20). In this embodiment, as shown in FIG. 3A, the injection is performed up to Y [mm]. Therefore, as shown to Fig.3 (a), the container main body 21 in the state by which the liquid mixture 10 was inject | poured has the space | gap part 11 in the upper part.

  In this embodiment, in a state where the container body 21 (manufacturing container 20) into which the mixed liquid 10 has been injected is left horizontally, the height of the container main body 21 is set to X [mm], and the mixed liquid 10 in the container main body 21 When the height of the liquid level is Y [mm], it is preferable that the relationship of 0.2 ≦ Y / X ≦ 0.7 is satisfied. By satisfying the above relationship, there is a sufficiently large gap 11, and in the step (S 4), the mixed solution 10 is vigorously collided with the upper and lower surfaces and side surfaces (particularly the upper and lower surfaces) of the production container 20. be able to. Due to this collision, a shock wave is generated in the mixed liquid 10, and the bubble 1 can be easily formed in the mixed liquid 10.

  In addition, as for the said relationship, it is more preferable to satisfy the relationship of 0.3 <= Y / X <= 0.5, and it is still more preferable to satisfy the relationship of 0.35 <= Y / X <= 0.4. Thereby, a bubble can be more easily formed in the liquid mixture 10 in a process (S4).

[S3] Step of sealing the production container Next, the container body 21 is filled with the gas 3, and the inside of the production container 20 is pressurized and sealed (see FIG. 3B). Specifically, after the gap 11 of the container body 21 into which the mixed liquid 10 has been injected is purged with the gas 3, the lid 22 is inserted into the opening (bottle opening) of the container body 21. Thereby, the liquid mixture 10 and the gas 3 are sealed in the manufacturing container 20.

Specifically, a syringe filled with gas 3 is prepared. Then, the rubber stopper 221 is punctured with the injection needle of the syringe. Thereafter, the gas 3 is further added into the production container 20 from the syringe. Thereby, the inside of the manufacturing container 20 is pressurized. Thereafter, by removing the injection needle from the lid 22 (rubber plug 221), the production container 20 sealed in a state where the inside of the production container 20 is pressurized by the gas 3 can be obtained.
As the gas 3, the various gases described above are used.

  In the bubble manufacturing method of the present invention, the pressure in the manufacturing container 20 (the pressure of the gas 3 filled in the gap portion 11) is made larger than 1.0 atm. In particular, the pressure in the production container 20 is preferably 1.5 to 10 atm, and more preferably 2 to 5 atm. Thereby, a part of the gas 3 is dissolved in the mixed liquid 10.

  When the gas 3 is dissolved in the mixed solution 10, the gas 3 in the mixed solution 10 is taken into the bubble 1 generated in the step (S4). Thereby, in process (S4), the bubble 1 can be produced | generated in large quantities in the liquid mixture 10. FIG.

  Moreover, the particle size and content of the bubble 1 produced | generated in the liquid mixture 10 can be adjusted by setting the pressure in the manufacturing container 20 to the arbitrary values larger than 1.0 atm.

[S4] Step of Vibrating Manufacturing Container Next, the manufacturing container 20 is vibrated so that the mixed liquid 10 repeatedly collides with the upper and lower surfaces and side surfaces (particularly the upper and lower surfaces) of the manufacturing container 20. In the present embodiment, as shown in FIG. 3C, the production container 20 is vibrated so that the production container 20 reciprocates substantially in the vertical direction.

  In this step, the manufacturing container 20 sealed in the step (S3) (the lower diagram in FIG. 3C) is vibrated upward (the middle diagram in FIG. 3C). Thereby, the liquid mixture 10 moves to the vicinity of the middle of the manufacturing container 20. When the manufacturing container 20 is further vibrated upward, the mixed solution 10 moves to the upper part of the manufacturing container 20 and collides with the lower surface (rubber plug 221) of the lid 22 (upper view in FIG. 3C). At this time, a shock wave is generated as shown in FIG. The pressure of the shock wave makes the outer material in the mixed liquid 10 a bubble 1. The bubble 1 contains the gas 3 dissolved in the mixed solution 10 and the gas 3 dissolved or mixed in the mixed solution 10 by vibration.

  On the other hand, the manufacturing container 20 (the upper diagram in FIG. 3C) is vibrated downward (the middle diagram in FIG. 3C). Thereby, the liquid mixture 10 moves to the vicinity of the middle of the manufacturing container 20. When the manufacturing container 20 is further vibrated downward, the mixed liquid 10 moves to the lower part of the manufacturing container 20 and collides with the lower surface of the manufacturing container 20 (the lower diagram in FIG. 3C). Also at this time, shock waves are generated as shown in FIG.

  Further, when the production container 20 is vibrated in the vertical direction, the mixed liquid 10 also collides with the inner side surface of the production container 20. Also at this time, shock waves are generated as shown in FIG.

  By repeating the above operations, a large amount of bubbles 1 of uniform size can be stably generated in the mixed liquid 10.

  In the bubble production method of the present invention, the production container 20 is vibrated at 5000 rpm or more in order to obtain a sufficiently fine and uniform-sized bubble 1. Thereby, the magnitude | size (pressure) of the shock wave generated when the liquid mixture 10 and the production container 20 collide becomes sufficiently large, the bubbles 1 generated in the liquid mixture 10 are miniaturized, and the size is made uniform. be able to. In addition, when the number of rotations of the manufacturing container 20 is set to a low value within the above range, the magnitude of the generated shock wave becomes small, so that the bubble 1 having a relatively large particle size can be generated. In addition, when the rotational speed is set high, the magnitude of the generated shock wave becomes large, so that the bubble 1 having a relatively small particle size can be generated.

  Although the rotation speed of the production container 20 is 5000 rpm or more, it is preferable that it is 5500 rpm or more, and it is more preferable that it is 6000-20000 rpm. By making the rotation speed of the production container 20 within the above range, it is possible to more reliably prevent the bubbles 1 generated by vibration from collapsing due to collision or coalescing and coarsening. Thereby, the bubble 1 of a more uniform size can be produced in a large amount in the mixed liquid 10 while reducing the particle diameter of the bubble 1.

  As an apparatus that can vibrate the production container 20 at the above rotation speed, for example, a bead type high-speed cell disruption system (homogenizer) can be used. As a specific example, Precellys manufactured by Bertin Technologies, Inc. can be used.

  Moreover, it is preferable that the pressure of the shock wave generated when the liquid mixture 10 and the production container 20 collide is 40 kPa to 1 GPa. When the pressure of the shock wave generated at the time of the collision between the mixed liquid 10 and the production container 20 falls within the above range, the bubbles 1 generated in the mixed liquid 10 can be made finer and the size thereof can be made more uniform. In particular, as the pressure of the shock wave generated at the time of the collision between the mixed liquid 10 and the production container 20 increases, the finer bubble 1 can be generated.

  When the manufacturing container 20 is vibrated, the vertical vibration width of the manufacturing container 20 is preferably about 0.7X to 1.5X [mm], more preferably about 0.8X to 1X [mm]. More preferred. Thereby, when the manufacturing container 20 vibrates, the liquid mixture 10 and the lower surface of the manufacturing container 20 and the lid 22 can be reliably collided, and the number of times of collision between the liquid mixture 10 and the lower surface of the manufacturing container 20 and the lid 22 is sufficient. Can be much more. Further, by vibrating the production container 20 with a sufficient vibration width in this way, the speed at which the mixed liquid 10 moves in the production container 20 is increased. Therefore, the magnitude of the collision wave generated when the mixed liquid 10 collides with the lower surface of the manufacturing container 20 and the lid 22 is sufficiently increased. As a result, a large amount of fine bubbles 1 can be generated in the mixed liquid 10.

  Further, when the production container 20 is reciprocated in the vertical direction, the production container 20 is preferably vibrated in the horizontal direction. Thereby, since the liquid mixture 10 also collides with the inner side surface of the manufacturing container 20, more vibration waves can be generated in the liquid mixture 10. The horizontal vibration width of the manufacturing container 20 is preferably about 0.3X to 0.8X [mm], and more preferably about 0.5X to 0.7X [mm]. Thereby, the effect mentioned above becomes more remarkable.

  Further, in this step, the manufacturing is performed so that the instantaneous relative speed between the manufacturing container 20 and the mixed liquid 10 in the manufacturing container 20 is 40 km / h or more when the mixed liquid 10 collides with the upper and lower surfaces and side surfaces of the manufacturing container 20. It is preferable to vibrate the container 20. Moreover, it is more preferable to vibrate the production container 20 so that the instantaneous relative speed is 50 km / h or more. By satisfying the above conditions, the pressure of the shock wave generated when the mixed liquid 10 and the production container 20 collide can be sufficiently increased. As a result, the bubbles 1 generated in the mixed liquid 10 can be made finer and the size thereof can be made more uniform.

  The time for vibrating the production container 20 under the above conditions is preferably about 10 to 120 seconds, and more preferably about 30 to 60 seconds. By setting the vibration time of the manufacturing container 20 within the above range, the number of times the mixed liquid 10 collides with the manufacturing container 20 is sufficiently increased, so that a large amount of bubbles 1 can be generated in the mixed liquid 10. In addition, the amount of the bubble 1 produced | generated in the liquid mixture 10 can be increased more by setting the vibration time of the manufacturing container 20 long within the said range.

  In addition, the particle size of the bubble 1 produced | generated in the liquid mixture 10 can be adjusted by changing the pressure of the gas 3 in the manufacturing container 20, and the rotation speed of the manufacturing container 20 within the range mentioned above. For example, microbubbles having a size of several microns can be obtained by vibrating the production container 20 at a rotational speed of 5000 to 6000 rpm in a state where the pressure in the production container 20 is 1.0 to 2 atm. Further, nanobubbles having a size of several tens to several hundreds of nanometers can be obtained by vibrating the production container 20 at a rotational speed of 6000 to 8000 rpm in a state where the pressure in the production container 20 is 1.2 to 4 atm.

  In the present embodiment, the manufacturing container 20 is vibrated so that the manufacturing container 20 reciprocates substantially in the vertical direction, but the method of vibrating the manufacturing container 20 is not limited to this. For example, the production container 20 may be vibrated so that the production container 20 rotates in the horizontal direction and / or the vertical direction. Even in this case, the mixed liquid 10 in the production container 20 repeatedly impacts the upper and lower surfaces and side surfaces of the production container 20 to generate shock waves. Even if such a vibration method is used, a uniform amount of bubbles 1 can be stably generated in a large amount in the liquid mixture 10.

[S5] Step of standing the production container After the production container 20 is vibrated under the above conditions, the production container 20 is left still (see FIG. 3D). Thereby, the bubble 1 (refer FIG. 1A) of uniform size can be stably manufactured in large quantities in the manufacturing container 20. FIG. At the same time, a production container 20 containing a large amount of uniform-sized bubbles 1 is obtained.

  In addition, it is preferable that the process (S2), the process (S3), and the process (S4) described above are performed so as to maintain the temperature of the mixed liquid 10 constant. Thereby, since the characteristic (viscosity etc.) of the liquid mixture 10 is stabilized in the bubble production process, the bubbles 1 having a uniform particle diameter can be stably generated in the liquid mixture 10. As a method for maintaining the temperature of the mixed liquid 10 constant, for example, a method of performing each of the above-described steps (S2) to (S4) in a glove box or a thermostat. In particular, in this embodiment, since the production container 20 is vibrated at a high speed in the step (S4), the production container 20 easily generates heat due to the collision between the mixed liquid 10 and the inner surface of the production container 20. However, it is possible to reliably prevent the temperature of the mixed liquid 10 from rising by vibrating the manufacturing container 20 in the thermostatic bath. As a result, the bubbles 1 having a uniform particle diameter can be more stably generated in the mixed liquid 10.

  Through the steps (S1) to (S5), the bubble 1 having an average particle size of about 10 nm to 1000 μm is manufactured.

  In the conventional bubble manufacturing method, a large-scale reflux device and various systems (tubes, nozzles, compressors, etc.) constituting the bubble manufacturing device are required. For this reason, it has been difficult to maintain a clean and sterile environment when producing bubbles used in the food and medical fields. On the other hand, in this invention, since the manufacturing container 20 for manufacturing a bubble is small enough, a large-scale system is not required. Furthermore, in the present invention, since the bubble 1 can be manufactured in a closed, clean and aseptic environment (sealed manufacturing container 20), it can be suitably used in the food field, the medical field, and the like.

  In addition, the bubble 1 obtained as mentioned above can exist stably in the liquid mixture 10. Therefore, the obtained bubble-containing container can be stored for a long time at room temperature. Specifically, it can be stored for a period of 6 to 24 months. Further, even after being stored for such a long period of time, since the stability of the bubble 1 in the mixed solution 10 is high, it is possible to use it without having to vibrate the bubble-containing container again. Moreover, since the manufacturing container 20 with a small capacity | capacitance can be used as a manufacturing container, the unit price of a bubble containing container can be suppressed. Therefore, the bubble-containing container obtained as described above has an advantage that it is easy to handle for medical institutions.

2. Method of Use The bubble-containing container obtained as described above is used for ultrasonic diagnosis of a patient.

  Specifically, first, the injection needle of the syringe is punctured into the rubber stopper 221 of the lid 22. Next, the bubble-containing mixed liquid is sucked from the bubble-containing container. Then, the injection needle is removed from the rubber stopper 221 and injected into the body from the patient's vein or the like. Thereafter, the bubble 1 is carried to the affected part by blood flow.

  At the time of ultrasonic diagnosis, an ultrasonic signal having a frequency and output adjusted so that the bubble 1 does not rupture is transmitted to the bubble 1 at the timing when the bubble 1 reaches the site to be diagnosed. Thereafter, a signal reflected from the bubble 1 (reflected echo) is received and processed to display an image of the blood vessel. Thereby, ultrasonic diagnosis can be performed.

  A known ultrasonic probe can be used as a device that receives the ultrasonic wave and the reflected wave from the bubble 1.

  The bubble-containing container obtained as described above can be applied to various fields in addition to the use of ultrasonic diagnosis. For example, the bubble 1 in the bubble-containing container obtained as described above has a bactericidal effect on water and food and has an effect of maintaining the freshness of the food. Furthermore, in the liquid containing the bubble 1, water, and oil (hydrophobic component), a large amount of oil can be mixed with water. Utilizing this effect, it is possible to cook while suppressing separation of moisture and oil in the food. Therefore, the obtained bubble-containing liquid mixture can be used in the food field.

  Moreover, in said description, by performing process (S1)-process (S5), the bubble 1 of uniform size (refer FIG. 1 (a)) can be stably manufactured in large quantities in the manufacturing container 20. FIG. it can. However, the bubble manufacturing method of this embodiment is not limited to this. For example, after step (S5), step (S4) and step (S5) may be repeated at least once. By repeatedly performing the step (S4) and the step (S5), the bubbles 1 having a uniform particle diameter can be generated more stably.

Second Embodiment
Next, a second embodiment of the bubble manufacturing method of the present invention will be described. The bubble 1 shown in FIG. 1B and FIG. 1C described above can be manufactured by the bubble manufacturing method of the present embodiment.

  Hereinafter, the bubble manufacturing method of the second embodiment will be described with a focus on differences from the bubble manufacturing method of the first embodiment, and description of similar matters will be omitted.

1. Bubble Production Method In the present embodiment, the target substance includes the outer material and the drug 4. That is, the bubble manufacturing method of the present embodiment is the same as that of the first embodiment described above except that the liquid mixture 10 adjusted in the step (S1) includes the drug 4 in addition to the outer shell material and the aqueous medium. This is the same as the bubble manufacturing method.

[S1] Preparatory Step In the present embodiment, the material (outer material), drug 4 and aqueous medium constituting the outer shell 2 of the bubble 1 are placed in a mixed solution preparation container (hereinafter simply referred to as “preparation container”), And a drug 4 are dissolved in an aqueous medium to prepare a mixed solution 10. That is, a predetermined amount of the shell material, the drug 4 and the aqueous medium are added to the preparation container, and then stirred to dissolve the shell material and the drug 4 in the aqueous medium. The order in which the outer shell material, the drug 4, and the aqueous medium are put into the preparation container is not particularly limited. As a method for dissolving the shell material and the drug 4 in an aqueous medium, for example, stirring with a stirring bar, ultrasonic treatment, or the like can be used.
The kind and content of the outer shell material and the aqueous medium are the same as those in the first embodiment.

  As the drug 4, the above-described genes, drugs and the like are used. The content of the drug 4 contained in the prepared mixed liquid 10 is preferably 0.1 to 50 wt%, and more preferably 20 to 50 wt%. Thereby, sufficient quantity of the medicine 4 can be included in the bubble 1 manufactured. As a result, it is possible to manufacture the bubble 1 having a better therapeutic effect on the affected area. In addition, when the drug 4 constitutes the outer shell 2 instead of the outer shell material, the mixed liquid 10 may not include the outer shell material.

  By using the mixed liquid 10 adjusted in this way, similarly to the first embodiment described above, by performing the steps (S2) to (S5), the bubbles 1 (of uniform size) ( 1 (b)) can be stably manufactured in large quantities. At the same time, a production container 20 containing a large amount of uniform-sized bubbles 1 is obtained.

  In the step (S4), the outer material in the mixed liquid 10 becomes the bubble 1 by vibrating the manufacturing container 20 and causing the mixed liquid 10 to collide with the upper and lower surfaces and side surfaces of the manufacturing container 20. The bubble 1 contains a gas 3 dissolved in the mixed solution 10, a gas 3 dissolved or mixed in the mixed solution 10 by vibration, and a drug 4. As a result, in the generated bubble 1, the drug 4 is enclosed in the outer shell 2 together with the gas 3, or is contained or adsorbed in the outer shell 2 itself.

2. Method of Use The bubble-containing container obtained as described above is used for ultrasonic therapy or ultrasonic diagnosis of a patient.

  Specifically, first, the injection needle of the syringe is punctured into the rubber stopper 221 of the lid 22. Next, the bubble-containing mixed liquid is sucked from the bubble-containing container. Then, the injection needle is removed from the rubber stopper 221 and injected into the body from the patient's vein or the like. Thereafter, the bubble 1 is carried to the affected part by blood flow.

  In the ultrasonic treatment, when the bubble 1 reaches the vicinity of the affected part, the bubble 1 is ruptured by irradiating ultrasonic waves. Thus, the drug 4 in the bubble 1 can be intensively administered to the affected area.

  Such bubble-containing liquid mixture is also effective when combined with ultrasonic treatment and ultrasonic diagnosis. Specifically, the reflected wave is monitored by irradiating the bubble 1 in the blood vessel with an ultrasonic wave having a frequency and intensity so that the bubble 1 does not burst. Thereby, the position of bubble 1 can be grasped correctly. When the bubble 1 reaches the vicinity of the target affected part, the bubble is ruptured by ultrasonic waves. As a result, the drug can be more accurately administered to the affected area.

  In addition, when using the component (for example, carbon dioxide etc.) with high solubility in the aqueous medium mentioned above as the gas 3, when the predetermined time passes after producing the bubble 1, the gas 3 in the outer shell 2 Dissolve in aqueous medium. Thereafter, when the gas 3 in the outer shell 2 is completely dissolved in the aqueous medium, only the drug 4 is sealed in the outer shell 2. That is, in such a case, the bubble 1 becomes a liposome or a micelle in which only the drug 4 is enclosed in the outer shell 2. The liposomes or micelles thus obtained can also be used as drugs.

At the time of ultrasound treatment, ultrasound output for rupturing the bubble 1 injected into the body, is preferably about ^{0.1~30W / cm 2, 0.5~10W / cm} 2 of about It is more preferable that By setting the output of the ultrasonic wave within the above range, the bubble 1 can be more reliably ruptured, and damage to normal cells around the affected area can be reliably prevented. When the ultrasonic output is within the above range, the ultrasonic irradiation time is preferably about 10 to 120 seconds, and more preferably about 30 to 60 seconds.

  In addition, the frequency of the ultrasonic wave applied during the ultrasonic treatment is preferably about 100 kHz to 10 MHz, and more preferably about 700 kHz to 1 MHz. By setting the frequency of the ultrasonic wave to be irradiated within the above range, the bubble can be ruptured with a lower ultrasonic output.

  Also by the bubble manufacturing method of the second embodiment, the same operation and effect as the bubble manufacturing method of the first embodiment are produced.

<Third Embodiment>
Next, a third embodiment of the bubble manufacturing method of the present invention will be described.

  FIG. 5 is a flowchart for explaining a third embodiment of the bubble manufacturing method of the present invention.

  Hereinafter, the bubble manufacturing method of the third embodiment will be described focusing on differences from the bubble manufacturing methods of the first and second embodiments, and the description of the same matters will be omitted.

  As shown in FIG. 5, the bubble manufacturing method of the present embodiment includes steps (S6) and (S7) after the steps (S1) to (S5) of the first embodiment described above. . Step (S6) is a step of changing the pressure in the production container. Step (S7) is a step of setting the number of rotations for vibrating the production container.

  In the bubble manufacturing method of the present embodiment, when the rotation speed is not changed in step (S7) (“NO” is selected in step (S7) in FIG. 5), the step (S4 ′) and the step are further performed. (S5 ′). On the other hand, when the rotational speed is changed in the step (S7) (“YES” is selected in the step (S7) in FIG. 5), the step (S8) and the step (S9) are further included. Furthermore, the bubble manufacturing method of the present embodiment includes a step (S10) of changing the pressure in the manufacturing container. Hereinafter, each process after a process (S6) is demonstrated one by one.

  In the present embodiment, the target substance may be only the outer material as in the first embodiment, or may include the outer material and the drug 4 as in the second embodiment.

[S6] Step of changing the pressure in the production container The pressure in the bubble-containing container (production vessel 20) after the step (S5) of the first embodiment described above is changed.

(1) When making the pressure in a manufacturing container higher than the pressure in process (S3) The inside of the manufacturing container 20 is pressurized like the process (S3) mentioned above. The gas 3 to be injected may be the same as or different from the gas 3 used in the step (S3), but the same gas 3 is used from the viewpoint of the stability of the finally generated bubbles. preferable.

  In such a manufacturing container 20, since the inside of the manufacturing container 20 is further pressurized more than the pressure in the manufacturing container 20 in a process (S3), the quantity of the gas 3 melt | dissolved in the liquid mixture 10 is process (S3). More than the amount of the gas 3 dissolved in the mixed liquid 10 in FIG. Therefore, when the manufacturing container 20 is vibrated again in the step (S4 ′) or the step (S8) described later, the gas 3 in the mixed liquid 10 is easily taken into the generated bubble 1, and as a result, the bubble 1 The production amount of increases. In addition, a pressure larger than the pressure applied to the bubble 1 generated in the step (S4) is applied to the mixed liquid 10. Thereby, since the bubble 1 of a production | generation process is compressed with a bigger pressure, the particle size of the bubble 1 tends to become small. Therefore, the bubble 1 with a smaller particle diameter than the bubble 1 produced | generated in a process (S4) can be produced | generated.

  In this case, the pressure in the production container 20 is preferably higher than the pressure in the step (S3) by 0.5 atm or more, more preferably higher by 1 to 10 atm. Thereby, the bubble 1 of a particle size smaller than the particle size of the bubble 1 produced | generated in the process (S4) mentioned above can be produced | generated more reliably.

(2) In the case where the pressure in the production container is made higher than 1.0 atm and lower than the pressure in step (S3) First, an empty syringe is prepared. Next, the rubber stopper 221 is punctured with an injection needle of the syringe, and the gas 3 in the manufacturing container 20 is sucked into the syringe. Thereby, the inside of the production container 20 is depressurized. Thereafter, the pressure in the manufacturing container 20 can be lowered by removing the injection needle from the rubber stopper 221.

  In such a manufacturing container 20, when the manufacturing container 20 is vibrated again in the step (S4 ′) or the step (S8) described later, the pressure applied to the mixed liquid 10 is the mixing generated in the step (S4). Less than the pressure applied to the liquid 10. Thereby, the extent to which the bubble 1 in the generation process is compressed becomes small, and the particle size of the bubble 1 tends to become large. Therefore, it is possible to generate bubbles 1 having a larger particle size than bubbles 1 generated in step (S4).

  In this case, the pressure in the manufacturing container 20 is appropriately adjusted within a range up to a pressure higher than 1.0 atm and lower than the pressure in the step (S3).

[S7] Step of setting the number of revolutions for vibrating the production container After changing the pressure in the production container 20 as described above, the number of revolutions for revibrating the container is set.

  The number of rotations when the container is re-vibrated is set to 5000 rpm or more as in the above-described step (S4).

  In the step (S7), when the number of rotations when the container is revibrated is not changed from the number of rotations in the step (S4), that is, when “NO” is selected in the step (S7) in FIG. (S4 ′) is performed.

  On the other hand, in the step (S7), when changing the rotation speed when the container is revibrated from the rotation speed in the step (S4), that is, when “YES” is selected in the step (S7) in FIG. A process (S8) is performed.

[S4 ′] Step of Revibrating the Production Container As described above, the production vessel 20 with the pressure changed is revibrated at the same rotational speed as in the step (S4) described above. Thereby, the bubble 1 of a particle size different from the bubble 1 produced | generated in a process (S4) is produced | generated in the liquid mixture 10. As shown in FIG.

  In this step, since the production container 20 is revibrated at the same rotational speed as in the step (S4), the particle size of the bubble 1 newly generated in the present embodiment changes according to the pressure changed in the step (S6). To do. That is, since the particle diameter of the bubble 1 can be adjusted by changing the pressure, the bubble 1 having a desired different particle diameter (average particle diameter) can be manufactured with good reproducibility. Moreover, since it is not necessary to change the setting of the apparatus which vibrates the manufacturing container 20, the bubble 1 which has a different particle size can be manufactured more simply.

[S5 ′] Step of Standing the Production Container After the production container 20 is vibrated under the above conditions, the production container 20 is allowed to stand in the same manner as in the step (S5). Thereby, the bubble 1 of a different size can be stably manufactured in large quantities in the manufacturing container 20. At the same time, a production container 20 (bubble-containing container) containing a large amount of such bubbles 1 is obtained.
A process (S10) is performed after standing still.

[S8] Step of re-vibrating the production vessel The production vessel 20 with the pressure changed is re-vibrated at a different rotational speed from the step (S4) described above. Thereby, the bubble 1 of a particle size different from the bubble 1 produced | generated in a process (S4) is produced | generated in the liquid mixture 10. As shown in FIG.

(1) When re-vibrating the production container at a higher rotational speed than in the step (S4) The above-described process (S4) except that the rotational speed at which the production container 20 is vibrated is higher than the rotational speed in the step (S4). The manufacturing container 20 is vibrated again in the same manner as in FIG.

  In this case, the rotation speed of the production container 20 is not particularly limited as long as it is higher than the rotation speed in the step (S4), but it is preferably 6000 to 20000 rpm, and more preferably 7000 to 20000 rpm. Thereby, since the manufacturing container 20 is vibrated at a rotational speed larger than the rotational speed in the step (S4), it is possible to generate the bubble 1 having a smaller particle diameter than the bubble 1 generated in the step (S4). Moreover, by making the rotation speed of the manufacturing container 20 within the above range, the bubbles 1 generated in the step (S4) and this step can be more reliably prevented from collapsing or coalescing to become coarse. can do. Thereby, the bubble 1 produced | generated in the process (S4) and the bubble 1 of a particle size smaller than the bubble 1 produced | generated in the process (S4) can be manufactured.

(2) In the case where the production container is revibrated at a lower rotational speed than in the step (S4), the above-described process (S4) except that the rotational speed at which the production container 20 is vibrated is lower than the rotational speed in the step (S4). The manufacturing container 20 is vibrated again in the same manner as in FIG.

  In this case, the rotational speed of the production container 20 is not particularly limited as long as it is lower than the rotational speed in the step (S4), but it is preferably 5000 to 9000 rpm, and more preferably 5500 to 7500 rpm. Thereby, since the manufacturing container 20 is vibrated at a rotational speed lower than the rotational speed in the step (S4), it is possible to generate the bubble 1 having a larger particle diameter than the bubble 1 generated in the step (S4). Moreover, by making the rotation speed of the manufacturing container 20 within the above range, the bubbles 1 generated in the step (S4) and this step can be more reliably prevented from collapsing or coalescing to become coarse. can do. Thereby, the bubble 1 produced | generated in the process (S4) and the bubble 1 of a particle size larger than the bubble 1 produced | generated in the process (S4) can be manufactured.

  In this step, since the production container 20 is revibrated at a different speed from that in the step (S4), the particle size of the bubble 1 newly generated in this embodiment is the pressure change in the production container 20 and the revibration. Varies depending on the number of rotations. In this way, by changing both the pressure in the manufacturing container 20 and the number of rotations at the time of re-vibration, it is possible to manufacture the bubble 1 having a particle size greatly different from the particle size obtained in the first embodiment described above. it can. Therefore, this process is advantageous when producing bubbles 1 having greatly different average particle diameters.

[S9] Step of standing the production container After the production container 20 is vibrated in the step (S8), the production container 20 is allowed to stand in the same manner as in the step (S5). Thereby, the bubble 1 of a different size can be stably manufactured in the manufacturing container 20. At the same time, a production container 20 (bubble-containing container) containing a large amount of such bubbles 1 is obtained.
A process (S10) is performed after standing still.

[S10] Step of changing pressure in manufacturing container again When the pressure in manufacturing container 20 is not changed, that is, when “NO” is selected in step (S10) in FIG. The method ends. Thereby, the 1st bubble 1 and the 2nd bubble 1 which each have a different average particle diameter in the range whose average particle diameter is 10 nm-1000 micrometers are manufactured.

  On the other hand, when the pressure in the manufacturing container 20 is changed, that is, when “YES” is selected in step (S10) in FIG. 5, step (S7) is performed. Thereafter, the above-described steps (S4 '), (S5') and (S10), or steps (S8), (S9) and (S10) are repeated. Thereby, the bubble 1 which has a some average particle diameter which is mutually different in the range whose average particle diameter is 10 nm-1000 micrometers can be manufactured.

  In the step (S10), when the pressure is repeatedly changed, the number of bubbles 1 having a plurality of different average particle diameters corresponding to the number of times the pressure is changed can be manufactured.

  The bubble-containing container obtained as described above contains bubbles 1 having different particle sizes in the mixed liquid 10. Due to the difference in size, the ease of passage of the bubble 1 into the blood vessel and the site to be transported also change (for example, the smaller the size of the bubble 1, the more it can be transported to the tip of the capillary). The bubble-containing liquid mixture thus obtained can be used in a multifaceted manner in accordance with the purpose of ultrasonic therapy.

  The bubble manufacturing method of the third embodiment also produces the same operations and effects as the bubble manufacturing methods of the first and second embodiments.

<Fourth embodiment>
Next, a fourth embodiment of the bubble manufacturing method of the present invention will be described.

FIG. 6 is a partial cross-sectional view showing the vicinity of the lid of the production container used in the fourth embodiment of the bubble production method of the present invention.
In the following description, the upper side in FIG. 6 is referred to as “upper”, and the lower side in FIG. 6 is referred to as “lower”.

  Hereinafter, the bubble manufacturing method of the fourth embodiment will be described focusing on the differences from the bubble manufacturing methods of the first to third embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of the present embodiment is the same as the bubble manufacturing method of the first to third embodiments described above except that the configuration of the lid 22 of the manufacturing container 20 is different.

  The lid 22 shown in FIG. 6 includes a bottom plate portion 223 provided on the lower surface of the rubber plug 221 in addition to the rubber plug 221 and the tightening portion 222 described above.

  The bottom plate portion 223 is a member having a disk shape smaller in diameter than the rubber plug 221. Further, the bottom plate portion 223 is formed with an opening 224 through which the injection needle of the syringe is inserted at a substantially center in plan view. Note that the size of the opening 224 is not particularly limited. However, when the opening 224 is the same size as the rubber plug 221 exposed from the tightening portion 222, the injection needle is punctured anywhere in the region exposed from the tightening portion 222 of the rubber plug 221 in the step (S3). can do. Moreover, as shown in FIG. 6, the opening 224 may be of a size that allows the injection needle to be inserted. In this case, a mark is attached to the upper surface of the rubber plug 221 at a position corresponding to the opening 224 (not shown), and an injection needle may be punctured into the rubber plug 221 using this mark as a mark.

Examples of the constituent material of the bottom plate portion 223 include various ceramic materials and various metal materials, and materials having a density higher than glass (2000 kg / m ³ or more) are preferable. Such materials, cast iron (density: 7000~7700kg / ^m about ^3), chrome nickel steel 18/8 (density: 7900kg / ^m about ^3), V2A steel (density: 7900kg / ^m about ³⁾ stainless steel or the like Steel, aluminum (density: about 2700 kg / m ³ ), geralumin (density: about 2700 kg / m ³ ), lead (density: about 11340 kg / m ³ ), iron (density: about 7870 kg / m ³ ), copper (density: 8900kg / ^{m 3} approximately), brass (density: 8250~8500kg / ^m about ^3), nickel (density: 8350kg / ^m about ^3), cast iron (density: 7000~7700kg / ^m about ^3), zinc (density: 7130kg / m ³ ), tin (density: about 7280 kg / m ³ ), etc., and one or more of these are combined Can be used. Among these materials, it is particularly preferable to use an iron alloy such as iron or stainless steel from the viewpoint of high specific gravity and high corrosion resistance to the constituent components of the mixed liquid 10.

  The bottom plate portion 223 made of such a material has a larger mass than a member formed using a material having a low specific gravity (density) such as synthetic rubber or resin. By increasing the mass of the bottom plate portion 223, the magnitude of the shock wave generated when the mixed liquid 10 collides with the upper surface (bottom plate portion 223) of the production container 20 in the step (S4) can be increased. As a result, the fine bubbles 1 can be more easily and stably generated in the mixed liquid 10.

  When such a lid 22 is used, in step (S3), the rubber plug 221 is punctured so that the injection needle of the syringe passes through the opening 224 of the bottom plate portion 223. And the inside of the manufacturing container 20 is pressurized by adding the gas 3 further into the manufacturing container 20 from a syringe similarly to this embodiment mentioned above. Thereafter, by removing the injection needle from the lid 22 (rubber plug 221), the production container 20 sealed in a state where the inside of the production container 20 is pressurized by the gas 3 can be obtained.

  A bubble-containing container can be obtained through the same steps as the bubble manufacturing method of the first to third embodiments described above using the manufacturing container 20 having such a configuration (the bubble manufacturing container of the present embodiment).

  The bubble manufacturing method of the fourth embodiment produces the same operations and effects as the bubble manufacturing method of the first to third embodiments.

<Fifth Embodiment>
Next, a fifth embodiment of the bubble manufacturing method of the present invention will be described.

  FIGS. 7A to 7F are cross-sectional views schematically showing another configuration of the container used in the fifth embodiment of the bubble manufacturing method of the present invention.

  In the following description, the upper side in FIGS. 7A to 7F is referred to as “upper”, and the lower side in FIGS. 7A to 7F is referred to as “lower”.

  Hereinafter, the bubble manufacturing method of the fifth embodiment will be described with a focus on differences from the bubble manufacturing methods of the first to fourth embodiments, and description of similar matters will be omitted.

  The bubble production method of the present embodiment is the same as the bubble production method of the first to fourth embodiments described above, except that the shape of the container (production container) is different.

  In the present embodiment, the manufacturing container 20 (the bubble manufacturing container of the present embodiment) is adapted to the shape of the container main body 21 having various shapes shown in FIGS. 7A to 7D and the shape of the upper surface of each container main body 21. And a lid (not shown). As shown in FIGS. 7A to 7D, the inner surface of the container body 21 includes at least one of a concave surface, a convex surface, and an uneven surface.

  A container body 21 shown in FIG. 7A is a container whose upper surface and lower surface are convex surfaces protruding inward. The production container 20 shown in FIG. 7B is a container whose upper and lower surfaces are uneven surfaces. A manufacturing container 20 shown in FIG. 7C is a container whose upper surface and lower surface are concave surfaces protruding outward. The manufacturing container shown in FIG. 7D is a container whose side surface is a convex surface that curves toward the inside of the manufacturing container 20.

  Such a surface (concave surface, convex surface, or uneven surface) has a larger surface area than a flat surface. Therefore, in the above-described step (S4), the area where the mixed liquid 10 can collide with the inner surface of the manufacturing container is increased, and more shock waves can be generated. Moreover, in such a surface, the degree of pressure of the generated shock wave varies depending on the shape. Thereby, a large amount of bubbles 1 having different particle diameters can be generated in the liquid mixture 10.

  Moreover, as shown in FIG.7 (e), the height of the container main body 21 can be made higher than the container main body 21 of 1st-4th embodiment mentioned above. When such a container main body 21 is used, the moving distance of the mixed liquid 10 becomes long when the manufacturing container is vibrated in the steps (S4, S4 'and S8). Therefore, the magnitude of the shock wave generated at the time of the collision can be increased. Thereby, the size of the bubble 1 manufactured in the liquid mixture 10 can be made smaller.

  On the other hand, the height of the container main body 21 can be made lower than the container main body 21 of the first to fourth embodiments described above, as shown in FIG. When such a container main body 21 is used, when the manufacturing container is vibrated in the steps (S4, S4 'and S8), the moving distance of the mixed liquid 10 is shortened. Therefore, since the frequency | count that the liquid mixture 10 collides with the inner surface of a manufacturing container can be increased, more pressure by the shock wave can be applied to the liquid mixture 10. Thereby, more bubbles 1 can be produced in the mixed liquid 10.

  Also by the bubble manufacturing method of the fifth embodiment, the same operation and effect as the bubble manufacturing method of the first to fourth embodiments are produced.

<Sixth Embodiment>
Next, a sixth embodiment of the bubble manufacturing method of the present invention will be described.

  FIGS. 8A to 8C are perspective views for explaining a sixth embodiment of the bubble manufacturing method of the present invention. FIG. 9 is a view for explaining a configuration (handle is omitted) in the vicinity of the rubber plug of the mini-nert valve shown in FIG. 8 (a), and FIG. FIG. 9B is a cross-sectional view taken along line XX of FIG. FIG. 10 is a cross-sectional view of the bubble-containing container shown in FIG.

  In the following description, the upper side in FIGS. 8A to 8C, FIG. 9B, and FIG. 10 and the front side in FIG. 9A are referred to as “up”, and FIG. ) To (c), FIG. 9 (b), the lower side in FIG. 10 and the back side in FIG. 9 (a) are referred to as “lower”. Further, the left side in FIGS. 8A to 8C is referred to as “left”, and the right side in FIGS. 8A to 8C is referred to as “right”.

  Hereinafter, the bubble manufacturing method of the sixth embodiment will be described focusing on the differences from the bubble manufacturing methods of the first to fifth embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of the present embodiment is the same as the bubble manufacturing method of the first to fifth embodiments described above except that the configuration of the manufacturing container is different.

[S1] Preparation Step A manufacturing container 20 (a bubble manufacturing container according to this embodiment) as shown in FIG. 8A is prepared.

  In the present embodiment, the manufacturing container 20 includes a container body 21 and a lid 22, similarly to the manufacturing container 20 of the first embodiment described above. As shown in FIG. 8A, the lid 22 includes a mininert valve 30 and a tube 33 that connects the rubber stopper 221 and the mininert valve 30 on the upper side thereof.

  The mini-nert valve 30 includes a valve main body 31 having a through hole 311, a rubber plug 32 embedded in the through hole 311 and formed with a pipe line 321 penetrating in the thickness direction (vertical direction), and opening and closing of the pipe line 321. And a handle 34 for controlling the control.

  The valve body 31 has a substantially rectangular parallelepiped shape, and a through-hole 311 that penetrates in the thickness direction (vertical direction) is formed near the center in the longitudinal direction (see FIGS. 9A and 9B). ). Further, as shown in FIG. 9B, the valve body 31 is formed with a through hole 312 penetrating in the longitudinal direction.

  The rubber plug 32 has a substantially cylindrical shape, and is made of, for example, silicon rubber. The rubber plug 32 is inserted into the through hole 311 of the valve body 31 and is fixed (embedded) in the valve body 31. In the approximate center of the rubber plug 32, a pipe line 321 is formed in which the injection needle 41 of the syringe 40 can be inserted. The rubber plug 32 is formed with a through hole 322 that is orthogonal to the pipe 321 and penetrates in the width direction (left-right direction) of the rubber plug 32 (see FIG. 9B). The through hole 322 of the rubber plug 32 and the through hole 312 of the valve body 31 communicate with each other, and a shaft 342 of the handle 34 described later is slidably inserted into the through hole 322 and the through hole 312.

  The handle 34 is connected to a pair of knobs 341 provided on both ends in the longitudinal direction (left and right direction) of the valve body 31 and the knobs 341, and is connected to the through hole 322 of the rubber plug 32 and the through hole 312 of the valve body 31. And a shaft 342 slidably inserted. A part of the shaft 342 is formed with a through hole 343 penetrating in the thickness direction (vertical direction in FIG. 8A).

  Although the tube 33 is not specifically limited, For example, it is comprised with the tube made from a silicon | silicone. The upper end portion of the tube 33 communicates with the pipe line 321 of the rubber plug 32, and the lower end portion of the tube 33 communicates with the internal space (gap portion 11) of the container main body 21 via the rubber plug 221. . In other words, the pipe line 321 and the internal space of the container main body 21 communicate with each other via the tube 33.

  In the manufacturing container 20 of the present embodiment, as shown in FIG. 8A, the left knob 341 is pushed to the right to bring the left knob 341 and the left end of the valve body 31 into contact with each other. In plan view (top view), the through hole 343 of the shaft 342 and the pipe line 321 overlap (communicate). Thereby, the pipe line 321 is opened, and the pipe line 321, the tube 33, and the internal space of the container main body 21 are communicated (see FIGS. 8A and 9B). On the other hand, as shown in FIG. 8B, the right knob 341 is pushed to the left to bring the right knob 341 and the right end of the valve body 31 into contact with each other. The position of the through hole 343 of the shaft 342 and the position of the pipe line 321 are shifted. Thereby, the pipe line 321 is closed and the inside of the manufacturing container 20 is sealed.

  Moreover, in this embodiment, the lid | cover 22 is provided with the rubber stopper 221, the fastening part 222, and the baseplate part 223 similarly to the lid | cover 22 shown in FIG. 6 (refer FIG. 10). As shown in FIG. 10, the lower end portion of the tube 33 (the end portion on the side connected to the rubber plug 221) is disposed at a position corresponding to the opening 224 of the bottom plate portion 223. Therefore, the tube 33 communicates with the inside of the container main body 21.

[S3] Step of sealing the production container After the gap 11 of the container body 21 into which the mixed liquid 10 has been injected is purged with the gas 3, the lid 22 is inserted into the opening (bottle opening) of the container body 21. Thereby, the liquid mixture 10 and the gas 3 are sealed in the manufacturing container 20.

  Next, a syringe 40 filled with the gas 3 is prepared. As shown in FIG. 8A, the knob 341 on the left side of the handle 34 is pressed to the right to open the pipe 321, and the pipe 321 and the tube 33 are communicated. Then, the injection needle 41 of the syringe 40 is inserted into the pipe line 321, and then the gas 3 is further added from the syringe 40 into the manufacturing container 20 through the mininert valve 30 and the tube 33. Thereby, the inside of the manufacturing container 20 is pressurized.

  Thereafter, the injection needle 41 is removed from the conduit 321 and simultaneously the knob 341 on the right side of the handle 34 is pressed to the left to close the conduit 321 so that the conduit 321 and the tube 33 are not in communication.

  Next, heat treatment is performed on a part of the tube 33 to form a sealing portion 331 in which the space in the tube 33 is closed (see FIG. 8B).

  Thereafter, the tube 33 is cut at the side of the sealing part 331 on the side of the mininert valve 30 (upper side in the drawing), and the manufacturing container 20 sealed in a state where the inside of the manufacturing container 20 is pressurized by the gas 3 can be obtained. (See FIG. 8 (c)).

  Thereafter, by performing steps (S4) and (S5) as in the first and second embodiments described above, or by performing steps (S4) to (S10) as in the third embodiment. A large amount of bubbles 1 of uniform size can be stably produced in the production container 20. At the same time, a production container (sealed container) 20 (bubble-containing container) containing a large amount of uniform-sized bubbles 1 is obtained.

  In the present embodiment, in the step (S3), the inside of the production container 20 can be pressurized without directly piercing the rubber stopper 221 of the lid 22 with the injection needle 41. That is, in this embodiment, since there is no through hole in the lid 22 and the tube 33 (section from the rubber plug 221 to the sealing portion 331), the airtightness in the manufacturing container 20 can be improved. By improving the airtightness in the manufacturing container 20, the bubble 1 can finally exist in the liquid mixture 10 more stably in the bubble-containing container finally obtained. That is, the long-term storage stability of the bubble-containing container is further improved.

  The bubble manufacturing method of the sixth embodiment produces the same operations and effects as the bubble manufacturing methods of the first to fifth embodiments.

<Seventh embodiment>
Next, a seventh embodiment of the bubble manufacturing method of the present invention will be described.

  FIG. 11: is a perspective view for demonstrating the manufacturing container used for 7th Embodiment of the manufacturing method of the bubble of this invention.

  In the following description, the upper side in FIG. 11 is referred to as “upper”, and the lower side in FIG. 11 is referred to as “lower”. Further, the left side in FIG. 11 is referred to as “left”, and the right side in FIG. 11 is referred to as “right”.

  Hereinafter, the bubble manufacturing method of the seventh embodiment will be described focusing on the differences from the bubble manufacturing methods of the first to sixth embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of the present embodiment is different from the bubble manufacturing method of the sixth embodiment described above in that the mini-nert valve 30 is provided on the side surface of the container body 21.

  As shown in FIG. 11, the manufacturing container 20 used in the present embodiment includes a container main body 21, a lid 22, a mini-nert valve 30, and a container main body 21 used in the bubble manufacturing method of the sixth embodiment described above. And a tube 33 for connecting the mini-nert valve 30 to each other. Further, in the manufacturing container 20 of the present embodiment, the mini-nert valve 30 is provided on the lid 22 side (upper side in the drawing) from the liquid level of the mixed liquid 10.

  The tube 33 is composed of a glass tube, and is formed integrally with the container body 21 (vial bottle).

  As shown in FIG. 11, the left end portion of the tube 33 communicates with the internal space (gap portion 11) of the container main body 21, and the right end portion of the tube 33 is connected to the conduit 321 of the rubber plug 32. Communicate. Therefore, also in this embodiment, the pipe line 321 and the internal space of the container main body 21 communicate with each other through the tube 33.

  Using the manufacturing container 20 having such a configuration, a bubble-containing container can be obtained through the same steps as the bubble manufacturing method of the sixth embodiment described above.

  The bubble manufacturing method of the seventh embodiment produces the same operations and effects as the bubble manufacturing method of the first to sixth embodiments.

<Eighth Embodiment>
Next, an eighth embodiment of the bubble manufacturing method of the present invention will be described.

  FIG. 12: is sectional drawing for demonstrating the manufacturing container used for 8th Embodiment of the manufacturing method of the bubble of this invention.

  In the following description, the upper side in FIG. 12 is referred to as “upper”, and the lower side in FIG. 12 is referred to as “lower”. Also, the left side in FIG. 12 is referred to as “left”, and the right side in FIG. 12 is referred to as “right”.

  Hereinafter, the bubble manufacturing method of the eighth embodiment will be described with a focus on differences from the bubble manufacturing methods of the first to seventh embodiments, and description of similar matters will be omitted.

  The bubble manufacturing method of this embodiment differs from the bubble manufacturing method of the first to third embodiments described above in that the configuration of the manufacturing container is different and the direction in which the manufacturing container is vibrated is different in step (S4). Is different. Specifically, in the step (S4), the manufacturing container 20 is vibrated so that the manufacturing container 20 shown in FIG. 12 reciprocates substantially in the horizontal direction.

[S1] Preparation Step A production container 20 (a container for producing bubbles according to this embodiment) as shown in FIG. 12 is prepared.

  The manufacturing container 20 of the present embodiment includes a container body 21 having a cylindrical portion 211 at the top, a rubber plug 23 that seals the opening of the cylindrical portion 211, and a weight portion 5 that is fixed to both ends of the container body 21. I have.

  The container body 21 has a substantially cylindrical shape that is long in the horizontal direction. A cylindrical portion 211 that protrudes vertically upward from the upper surface of the container body 21 is formed at the approximate center of the container body 21. In the container body 21 of the present embodiment, only the opening of the cylindrical portion 211 is open to the outside. In addition, screw grooves 212 are formed on both ends of the container body 21.

  The rubber plug 23 is not particularly limited. For example, a rubber plug made of silicon can be used.

  The weight portion 5 includes a flat plate portion 51 having a disk shape and a cylindrical portion 52 erected from an edge portion of the flat plate portion 51, and a member having a substantially C shape when the outer shape thereof is viewed in cross section. It is. A thread groove 521 that can be screwed into the thread groove 212 of the container body 21 is formed on the inner peripheral side of the cylindrical portion 52. By screwing the screw groove 521 of the weight part 5 into the screw groove 212 of the container body 21, the weight part 5 is attached to the container body 21 with the flat plate part 51 in close contact with each end of the container body 21 (fixed). )

  Similar to the bottom plate portion 223 of the lid 22 shown in FIG. 6 described above, the weight portion 5 is made of a material having a large specific gravity such as a ceramic material or a metal material. Therefore, by attaching the weight part 5 to the container main body 21, the mass of the both ends of the container main body 21 can be increased. Thereby, in the step (S4), the magnitude of the shock wave generated when the mixed liquid 10 collides with the portions (particularly both end portions) fixed to the weight portion 5 of the container body 21 can be increased. As a result, the fine bubbles 1 can be more easily and stably generated in the mixed liquid 10.

  In addition, as a constituent material of the weight part 5, iron or an iron alloy such as stainless steel is used among metal materials from the viewpoint of high specific gravity and high corrosion resistance to the constituent components of the mixed liquid 10. Particularly preferred.

  Moreover, such a weight part 5 can be easily attached to and detached from the container main body 21. By changing the constituent material and / or size of the weight part 5, the mass of the weight part 5 can be adjusted as appropriate. By adjusting the mass of the weight part 5, the size and generation amount of the bubbles 1 generated in the mixed liquid 10 can be adjusted in the step (S4). That is, in the bubble manufacturing method of the present embodiment, bubbles 1 having various sizes and contents can be manufactured using the same manufacturing container 20. Therefore, since it is not necessary to prepare a plurality of types of production containers 20 having different sizes in accordance with the target size and content of the bubble 1, the productivity of the bubble-containing container is improved.

[S3] Step of Sealing the Manufacturing Container After purging the inside of the container main body 21 into which the mixed liquid 10 has been injected with the gas 3, the rubber plug 23 is inserted into the opening of the cylindrical portion 211 of the container main body 21. Thereby, the liquid mixture 10 and the gas 3 are sealed in the manufacturing container 20.

  Next, a syringe 40 filled with the gas 3 is prepared. Then, the injection needle 41 of the syringe 40 is punctured into the rubber plug 23. Thereafter, the gas 3 is further added from the syringe 40 into the production container 20. Thereby, the inside of the manufacturing container 20 is pressurized. Thereafter, by removing the injection needle from the rubber stopper 23, it is possible to obtain the sealed manufacturing container 20 with the inside of the manufacturing container 20 being pressurized by the gas 3.

[S4] Step of Vibrating Manufacturing Container Next, the manufacturing container 20 is vibrated so that the mixed liquid 10 repeatedly collides with both ends and side surfaces (particularly both ends) of the manufacturing container 20. In the present embodiment, the manufacturing container 20 is vibrated so that the manufacturing container 20 reciprocates substantially in the horizontal direction (longitudinal direction) of the manufacturing container 20.

  In addition, the vibration of the manufacturing container 20 in this embodiment can be performed on the same conditions as the process (S4) of 1st Embodiment mentioned above.

  Thereafter, as in the first and second embodiments described above, the production container 20 is obtained by performing the step (S5) or by performing steps (S5) to (S10) as in the third embodiment. It is possible to stably produce a large amount of uniform-sized bubbles 1 inside. At the same time, a production container 20 containing a large amount of uniform-sized bubbles 1 is obtained.

  The bubble manufacturing method of the eighth embodiment produces the same operations and effects as the bubble manufacturing method of the first to seventh embodiments.

<Ninth Embodiment>
Next, a ninth embodiment of the bubble manufacturing method of the present invention will be described.

  FIG. 13: is sectional drawing for demonstrating the manufacturing container used for 9th Embodiment of the manufacturing method of the bubble of this invention. FIG. 13A shows the manufacturing container in an exploded state, and FIG. 13B shows the manufacturing container in an assembled state.

  In the following description, the upper side in FIGS. 13A and 13B is referred to as “upper”, and the lower side in FIGS. 13A and 13B is referred to as “lower”. Further, the left side in FIGS. 13A and 13B is referred to as “left”, and the right side in FIGS. 13A and 13B is referred to as “right”.

  Hereinafter, the bubble manufacturing method according to the ninth embodiment will be described focusing on the differences from the bubble manufacturing methods according to the first to eighth embodiments, and description of similar matters will be omitted.

  As shown in FIGS. 13A and 13B, the bubble manufacturing method of the present embodiment is the same as that of the eighth embodiment described above except that both ends of the container body 21 of the manufacturing container 20 are opened to the outside. 12 is the same as the bubble manufacturing method using the manufacturing container 20 shown in FIG. That is, the container main body 21 is configured by a cylindrical member having both ends opened to the outside.

  When using the manufacturing container 20 having such a configuration, first, the weight part 5 is attached to the container body 21 by screwing the thread groove 521 of the weight part 5 into the screw groove 212 of the container body 21. Note that, as shown in FIG. 13B, in a state where the weight portion 5 is attached to the container main body 21, the flat plate portion 51 is in close contact with each end of the container main body 21.

  Although not shown, a packing for improving the adhesion between the weight portion 5 and the container body 21 may be disposed between the flat plate portion 51 and each end of the container body 21. Thereby, the airtightness of the manufacturing container 20 can be improved.

  Then, a bubble containing container can be obtained through the process similar to the manufacturing method of the bubble of this embodiment mentioned above.

  The bubble-containing container thus obtained can be used after the rubber stopper 23 is punctured with an injection needle of a syringe and the bubble-containing mixed solution is sucked. Moreover, in this structure, the weight part 5 can be removed from the container main body 21, and a bubble containing liquid mixture can be taken out from a bubble containing container directly, without using a syringe.

  The bubble manufacturing method of the ninth embodiment produces the same operations and effects as the bubble manufacturing method of the first to eighth embodiments.

<Tenth Embodiment>
Next, a tenth embodiment of the bubble manufacturing method of the present invention will be described.

  FIG. 14: is sectional drawing for demonstrating the manufacturing container used for 10th Embodiment of the manufacturing method of the bubble of this invention. FIG. 14A shows the manufacturing container in an exploded state, and FIG. 14B shows the manufacturing container in an assembled state. FIG. 15 is a view for explaining the position of the opening formed in the lid of the manufacturing container shown in FIG. FIG. 15 (a) is a view for explaining a state before the injection needle of the syringe is punctured into the rubber stopper, and FIG. It is a figure for demonstrating the state fastened to the part.

  In the following description, the upper side in FIGS. 14A and 14B is referred to as “upper”, and the lower side in FIGS. 14A and 14B is referred to as “lower”. Further, the left side in FIGS. 14A and 14B is referred to as “left”, and the right side in FIGS. 14A and 14B is referred to as “right”.

  Hereinafter, the bubble manufacturing method of the tenth embodiment will be described with a focus on differences from the bubble manufacturing methods of the first to ninth embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of this embodiment is the same as the bubble manufacturing method of the first to third embodiments described above, except that the configuration of the lid of the manufacturing container is different.

[S1] Preparation Step A manufacturing container 20 as shown in FIG. 14B is prepared.

  The manufacturing container 20 of this embodiment has the same container main body 21 and the cover 22 as the manufacturing container 20 of 1st Embodiment mentioned above.

  In the present embodiment, the lid 22 includes a bottom plate portion 223 fixed to the bottle mouth of the container body 21, a rubber plug 221 disposed on the opposite side of the bottom plate portion 223 from the container body 21, and the rubber plug 221 as the bottom plate portion. And a tightening portion 222 that is fixed to 223.

  The bottom plate portion 223 has a flat plate portion 225 having a disc shape and a cylindrical portion 226 standing from the edge of the flat plate portion 225, and a member having a substantially C shape when the outer shape thereof is viewed in cross section. It is. In the present embodiment, the shape of the bottom plate portion 223 (flat plate portion 225) in plan view (top view) and the shape of the rubber plug 221 are substantially the same and have substantially the same diameter. In addition, thread grooves formed so as to be screwable with each other are formed on the inner peripheral surface of the cylindrical portion 226 and the outer peripheral surface on the bottle mouth side of the container main body 21, respectively, and by screwing these together, The bottom plate portion 223 (the flat plate portion 225) is fixed in a state of being in close contact with the bottle mouth of the container body 21.

  The bottom plate 223 is formed with an opening 224 having a size that allows the injection needle of the syringe to be inserted at a position spaced apart from the center of the bottom plate 223 in plan view (top view). That is, as shown in FIG. 15A, the center C of the rubber plug 221 and the opening 224 of the bottom plate portion 223 are misaligned in the plan view of the lid 22.

  Similar to the bottom plate portion 223 of the lid 22 shown in FIG. 6 described above, the bottom plate portion 223 is made of a material having a large specific gravity such as a ceramic material or a metal material. By increasing the mass of the bottom plate portion 223, the magnitude of the shock wave generated when the mixed liquid 10 collides with the upper surface (bottom plate portion 223) of the production container 20 in the step (S4) can be increased. As a result, the fine bubbles 1 can be more easily and stably generated in the mixed liquid 10.

  As the rubber plug 221, a rubber plug similar to the rubber plug 221 used in the bubble manufacturing method of the first embodiment described above can be used. On the surface of the rubber plug 221, a mark X for puncturing the injection needle is attached at a position corresponding to the opening 224 of the bottom plate portion 223 when the lid 22 is attached to the container main body 21 (FIG. 15). (See (a)).

  The tightening portion 222 is configured to cover the edge of the rubber plug 221. Further, screw grooves formed so as to be able to be screwed to each other are formed on the outer peripheral surfaces of the tightening portion 222 and the bottom plate portion 223 (flat plate portion 225), and the rubber plugs 221 are screwed together. Are fixed in close contact with the bottom plate portion 223 (flat plate portion 225).

[S3] Step of sealing the production container First, the gap 11 of the container body 21 into which the mixed liquid 10 has been injected is purged with the gas 3, and then the lid 22 is inserted into the opening (bottle opening) of the container body 21. (The state shown in FIG. 15A). Thereby, the liquid mixture 10 and the gas 3 are sealed in the manufacturing container 20.

  In the state shown in FIG. 15A, the injection needle of the syringe is pierced through the mark X of the rubber plug 221 and the injection needle is inserted through the opening 224 of the bottom plate portion 223. Then, the gas 3 is further added from the syringe into the production container 20 to pressurize the production container 20, and then the injection needle is removed from the rubber stopper 221.

  Next, the tightening portion 222 is turned to tighten the tightening portion 222 to the bottom plate portion 223 (the state shown in FIG. 15B). By tightening the tightening portion 222 to the bottom plate portion 223, the rubber plug 221 is compressed toward the bottom plate portion 223 while rotating (for example, rotating 180 °) with respect to the bottom plate portion 223. Therefore, in plan view, the position of the through hole 227 formed in the rubber stopper 221 and the position of the opening 224 of the bottom plate part 223 are shifted by puncture of the injection needle (see FIG. 15B). Thereby, the opening 224 of the bottom plate part 223 is closed by the rubber plug 221, and the manufacturing container 20 sealed in a state where the inside of the manufacturing container 20 is pressurized by the gas 3 can be obtained.

  In this embodiment, since there is no portion that communicates the inside and the outside of the manufacturing container 20, the hermeticity in the manufacturing container 20 can be improved. By improving the airtightness in the manufacturing container 20, the bubble 1 can finally exist in the liquid mixture 10 more stably in the bubble-containing container finally obtained. That is, the long-term storage stability of the bubble-containing container is further improved.

As described above, by tightening the tightening portion 222 to the bottom plate portion 223, the rubber plug 221 is compressed to the bottom plate portion 223 side. The thickness of the rubber plug 221 before the tightening portion 222 is tightened to the bottom plate portion 223 is t ₁ (mm), and the thickness of the rubber plug 221 when the tightening portion 222 is tightened to the bottom plate portion 223 is t _2. When it is (mm), the compression rate ((t ₁ -t ₂ ) / t ₁ × 100) of the rubber plug 221 is preferably 5 to 60%, and more preferably 10 to 30%. Thereby, the adhesiveness between the rubber plug 221 and the bottom plate portion 223 can be further improved while suppressing the load on the rubber plug 221 due to the tightening of the tightening portion 222. As a result, the airtightness in the manufacturing container 20 can be further improved.

  Also in this embodiment, a large amount of bubbles 1 of uniform size can be stably produced in the production container 20. At the same time, a production container 20 (bubble-containing container) containing a large amount of uniform-sized bubbles 1 is obtained.

  Also by the bubble manufacturing method of the tenth embodiment, the same operation and effect as the bubble manufacturing method of the first to ninth embodiments are produced.

As mentioned above, although the manufacturing method of the bubble of this invention and the container for bubble manufacture were demonstrated based on embodiment of illustration, this invention is not limited to this, Each process can exhibit the same function. Any step can be substituted.
For example, the arbitrary configurations of the first to tenth embodiments can be combined.

  Here, in order to elucidate the influence of the volume of the gas 3 enclosed in the production container 20 and the rotation speed of the production container 20 on the particle size and content of the bubbles 1 generated in the mixed liquid 10, The experiment was conducted.

  First, the relationship between the rotation speed of the production container 20 and the particle diameter and content of the bubbles 1 generated in the mixed liquid 10 was examined.

(Bubble manufacturing method)
[Preparation process]
First, 120 μl of an albumin solution containing 25 mg / ml of albumin (Albminer 25% manufactured by CSL Behring) was prepared, and 12 ml of 25% phosphate buffered saline was prepared. In addition, a 15 ml vial (height X: 50 mm, outer diameter R: 25 mm) was prepared. This vial has the same shape as the manufacturing container 20 shown in FIG.

[Step of injecting the mixed solution into the container]
The whole amount of albumin solution and 25% phosphate buffered saline was injected into the prepared vial. The liquid surface height Y of the mixed solution of albumin solution and 25% phosphate buffered saline was 25 mm.

[Process for sealing the container]
Next, after the space in the vial bottle into which the mixed liquid was injected was purged with perfluorobutane, a lid having the same shape as the lid 22 shown in FIG. 3 was inserted into the vial mouth of the vial bottle. Next, a syringe filled with perfluorobutane was prepared. An additional 2 ml of perfluorobutane was added from the syringe into the vial by puncturing a rubber stopper on the lid with a syringe needle. As a result, the vial was sealed with the pressure in the vial set to 2 atm to obtain a sealed vial.

[Step of vibrating container]
Next, two sealed vials were prepared. One sealed vial was vibrated at 5000 rpm for 30 seconds, and the other sealed vial was vibrated at 6500 rpm for 30 seconds, using Precellys (high-speed cell disruption system) manufactured by Bertin Technologies. At that time, the closed vial reciprocated in the vertical direction, and it was confirmed that the mixed solution repeatedly collided with the upper and lower surfaces of the vial. When the closed vial was vibrated, the vertical vibration width of the closed vial was 40 mm, and the horizontal vibration width of the closed vial was 20 mm.

[Step of leaving the container stationary]
After vibration, the sealed vial was allowed to stand to obtain a bubble-containing container.

(Particle size distribution measurement)
From the bubble-containing container obtained as described above, a liquid mixture containing bubbles (bubble-containing liquid mixture) was taken out with a syringe, and then mixed with a bubble measuring device (nanoparticle analysis system nanosight). The particle size distribution of the bubbles contained in was measured. The result is shown in FIG.

  FIG. 16A is a graph showing the particle size distribution of the bubbles when the bubbles are produced at a rotational speed of 5000 rpm and 6500 rpm. FIG.16 (b) is the elements on larger scale whose horizontal axis is the range of 0-700 nm in the graph shown to Fig.16 (a).

  As shown in FIG. 16 (a), the bubble content in the liquid mixture may be greatly increased by vibrating the sealed vial at 6500 rpm, compared to when the sealed vial is vibrated at 5000 rpm. did it. In particular, the content of bubbles having a particle size smaller than about 600 nm is 3 to 5 times higher when the closed vial is vibrated at 6500 rpm than when the closed vial is vibrated at 5000 rpm. became.

  Even when the number of rotations of the sealed vial is 5000 rpm, the bubble content in the mixed solution can be increased to some extent by increasing the vibration time. However, it is less than the bubble content when the rotational speed is 6500 rpm.

  In addition, as shown in FIG. 16B, when the closed vial was vibrated at 6500 rpm, a large amount of extremely small bubbles having a particle size of about 100 to 150 nm could be generated.

  The above results are considered to be due to the following actions and effects. That is, the magnitude of the pressure of the shock wave generated when the mixed solution collides with the vial changes according to the number of rotations of the closed vial. The magnitude of the pressure of the shock wave is a major factor that determines the particle size and content of bubbles generated in the mixed liquid. In the case of stirring using a general stirrer or vibration at a rotational speed smaller than 5000 rpm, such a shock wave does not occur or even if it occurs, the generated amount is small. For this reason, bubbles having a sufficiently small particle diameter as in the present invention cannot be generated in the liquid mixture with a sufficient content.

FIG. 17 shows the result of analyzing the particle size distribution graph shown in FIG.
FIG. 17A is a graph showing the relationship between the number of rotations of the closed vial and the average particle diameter of the bubbles. FIG. 17B is a graph showing the relationship between the rotational speed of the closed vial and the bubble content.

As shown in FIG. 17A, when the sealed vial is vibrated at 6500 rpm, the average particle size of the generated bubbles is about 80 nm smaller than that when the sealed vial is vibrated at 5000 rpm. Become. In addition, as shown in FIG. 17B, when the sealed vial is vibrated at 6500 rpm, the content of the generated bubbles is 9 × compared to the case where the sealed vial is vibrated at 5000 rpm. Decrease by about 10 ⁷ particles / ml. From this result, it can be seen that when the closed vial is vibrated at 6500 rpm, a larger amount of bubbles having a small particle diameter can be generated than when the closed vial is vibrated at 5000 rpm.

  Next, the relationship between the volume of the gas 3 sealed in the production container 20 and the particle size and content of the bubbles 1 generated in the mixed solution 10 was examined.

(Bubble manufacturing method)
In the step of sealing the container of Example 1, except that four sealed vials were prepared in which the volume of perfluorobutane sealed in the vial was changed to 0.5 ml, 1 ml, 1.5 ml, and 2 ml, respectively. A bubble-containing container was obtained in the same manner as in Example 1.

  For each sealed vial, the volume (ml) of gas to be sealed (perfluorobutane), the pressure in the sealed vial (atm), and the number of rotations (rpm) of the sealed vial in the step of vibrating the container are as follows: Table 1 shows.

(Particle size distribution measurement)
In the same manner as in Example 1, the particle size distribution measurement of the bubble-containing mixed solution in each obtained bubble-containing container was performed. The result of analyzing the graph of the obtained particle size distribution is shown in FIG.

  FIG. 18 (a) is a graph showing the relationship between the volume of gas sealed in a closed vial and the average particle diameter of bubbles. FIG. 18B is a graph showing the relationship between the volume of gas sealed in the closed vial and the bubble content.

  As shown in FIG. 18 (a), even when the number of rotations for vibrating the closed vial was the same, the average particle size of the generated bubbles was reduced by increasing the pressure in the closed vial. Specifically, when the pressure in the closed vial is 2 atm, the average particle diameter of the generated bubbles is about 100 nm smaller than that in the case where the pressure in the closed vial is 1.2 atm. It was.

  Moreover, as shown in FIG.18 (b), content of the bubble increased by making the pressure in an airtight vial bottle large. In particular, when the pressure in the closed vial was 2 atm, the bubble content was more than twice as large as that in the case where the pressure in the closed vial was 1.2 atm.

  By increasing the pressure in the closed vial, it is considered that the pressure of the shock wave generated when the mixed solution collides with the vial greatly affects the particle size and content of the generated bubbles. Therefore, even if the rotation speed which vibrates a closed vial is the same, the average particle diameter of the produced | generated bubble changes according to the pressure in a closed vial. Moreover, a large amount of gas is taken into the liquid mixture by increasing the pressure in the closed vial. For this reason, the content of bubbles generated in the mixed liquid can be increased by the action of the shock wave as described above.

(Method for producing nanobubbles containing GFP gene)
[Preparation process]
First, as in the first embodiment described above, 120 μl of albumin solution (CSL Behring's albumin 25%) and 12 ml of 25% phosphate buffered saline were prepared. Furthermore, 2 μg of GFP gene was prepared. In addition, a 15 ml vial (height X: 50 mm, outer diameter R: 25 mm) was prepared. This vial has the same shape as the manufacturing container 20 shown in FIG.

[Step of injecting the mixed solution into the container]
The albumin solution, 25% phosphate buffered saline, and GFP gene were all injected into the prepared vial. The liquid surface height Y of the mixed solution of albumin solution, 25% phosphate buffered saline and GFP gene was 25 mm.

[Process for sealing the container]
Next, after the space in the vial bottle into which the mixed liquid was injected was purged with perfluorobutane, a lid having the same shape as the lid 22 shown in FIG. 3 was inserted into the vial mouth of the vial bottle. Next, a syringe filled with perfluorobutane was prepared. An additional 2 ml of perfluorobutane was added from the syringe into the vial by puncturing a rubber stopper on the lid with a syringe needle. As a result, the vial was sealed with the pressure in the vial set to 2 atm to obtain a sealed vial.

[Step of vibrating container]
Next, the above-mentioned sealed vial was vibrated at a rotational speed of 7000 rpm for 30 seconds using Precellys manufactured by Bertin Technologies. At that time, the closed vial reciprocated in the vertical direction, and it was confirmed that the mixed solution repeatedly collided with the upper and lower surfaces of the vial. When the closed vial was vibrated, the vertical vibration width of the closed vial was 40 mm, and the horizontal vibration width of the closed vial was 20 mm.

[Step of leaving the container stationary]
After vibration, the sealed vial was allowed to stand to obtain a bubble-containing container. In addition, the liquid mixture (bubble containing liquid mixture) containing a bubble was taken out with the syringe, and the size of the bubble was confirmed using the bubble measuring apparatus (nanoparticle analysis system nanosight). As a result, the average particle size of the bubbles was 600 nm.

<Evaluation of introduction of fluorescent protein expression gene into cells>
Cerebrovascular pericytes (pericite) (product code: C-12980, manufactured by Takara Bio Inc.) was added to the petri dish with 0.2 μg of the mixed solution obtained in Example 3 to obtain cerebral vascular pericytes. A medium was obtained. Note that perisite is known as a cell in which gene transfer is very difficult.

  Four samples of such a cerebral vascular pericyte medium were prepared. These samples were irradiated with ultrasonic waves having a frequency of 1.0 MHz (sine wave, pulse repetition frequency (PRF): 100 Hz, duty ratio (DC): 10%) for 60 seconds with the following output.

[Irradiation output]
^{0.6W / cm 2, 0.8W / cm} 2, 0.9W / cm 2, 1.0W / cm 2
Thereafter, each sample after culturing the cerebral vascular pericyte culture medium at 37 ° C. for 48 hours was observed with a fluorescence microscope.

FIG. 19 is a fluorescence microscope image of a cerebral vascular pericyte cell culture medium cultured at 37 ° C. for 48 hours, and FIG. 19A is an image of a sample irradiated with ultrasound at an irradiation intensity of 0.6 W / cm ^2. FIG. 19B is an image of a sample irradiated with ultrasonic waves at an irradiation intensity of 0.8 W / cm ² . FIG. 20 is a fluorescence microscopic image of a cerebral vascular pericyte cell culture medium cultured at 37 ° C. for 48 hours, and FIG. 20 (a) shows a sample subjected to ultrasonic irradiation at an irradiation intensity of 0.9 W / cm ² . FIG. 20B is an image of a sample irradiated with ultrasonic waves at an irradiation intensity of 1.0 W / cm ² .

  As shown in FIGS. 19 (a), 19 (b) and 20 (a), 20 (b), a green colored region was confirmed in the sample irradiated with ultrasonic waves at any irradiation output. This indicates that green fluorescent protein (GFP) is expressed in the cerebral vascular pericytes in each sample. Therefore, in any sample, it was shown that the bubble burst by ultrasonic irradiation, and the GFP gene contained in the bubble was introduced into the brain pericytes.

  DESCRIPTION OF SYMBOLS 1 ... Bubble 2 ... Outer shell 3 ... Gas 4 ... Drug 10 ... Mixed liquid 11 ... Gap part 20 ... Manufacturing container 21 ... Container main body 211 ... Cylindrical part 212 ... Screw groove 22 ... Lid 221 ... Rubber stopper 222 ... Fastening part 223 ... Bottom plate part 224 ... Opening part 225 ... Flat plate part 226 ... Cylindrical part 227 ... Through hole 23 ... Rubber plug 30 ... Mininert valve 31 ... Bubble body 311, 312 ... Through hole 32 ... Rubber plug 321 ... Pipe line 322 ... Through hole 33 ... Tube 331 ... Sealing part 34 ... Handle 341 ... Knob 342 ... Shaft 343 ... Through hole 40 ... Syringe 41 ... Injection needle 5 ... Weight part 51 ... Flat plate part 52 ... Cylindrical part 521 ... Screw groove X ... Mark C ... Center

Claims (17)

Injecting a solution containing the target substance to a predetermined height of the container;
Vibrating the container at a rotational speed of 5000 rpm or more so that the solution repeatedly collides with the inner surface of the container in a state where the pressure in the container is greater than 1.0 atm,
The step of vibrating the container includes the step of vibrating the container by a rotational motion or a rotational motion and a reciprocating motion of the container in a vertical direction,
When the height of the container is X (mm), the vibration width in the vertical direction of the container is 0.7X to 1.5X (mm).   The bubble manufacturing method according to claim 1, wherein the step of vibrating the container includes a step of generating a shock wave by causing the solution to collide with the inner surface of the container.   The bubble production method according to claim 2, wherein the pressure of the shock wave is 40 kPa to 1 GPa. Vibrating the container further includes vibrating the container by a reciprocating and / or rotating movement of the container in a horizontal direction;
The bubble manufacturing method according to any one of claims 1 to 3, wherein the horizontal vibration width of the container is 0.3X to 0.8X (mm).   The step of vibrating the container is performed such that an instantaneous relative speed between the container and the solution when the solution collides with the inner surface of the container is 40 km / h or more. The manufacturing method of the bubble as described in 2.   The bubble manufacturing method according to claim 1, wherein the step of vibrating the container is performed in a state where the pressure is set to 1.5 to 10 atm.   The predetermined height is 0.2 ≦ Y /, where Y (mm) is the height of the liquid level of the solution in the container in a state where the container into which the solution has been poured is left to stand horizontally. The method for producing bubbles according to any one of claims 1 to 6, which satisfies a relationship of X≤0.7.   The bubble production according to any one of claims 1 to 7, further comprising a step of revibrating the container at a rotation speed of 5000 rpm or more after changing the pressure in the container after the step of vibrating the container. Method.   The bubble manufacturing method according to claim 8, wherein the step of vibrating the container again is performed such that the pressure is 1 to 10 atm higher than the pressure in the step of vibrating the container.   The bubble manufacturing method according to claim 8 or 9, wherein the step of revibrating the container is performed at a rotational speed different from the rotational speed in the step of vibrating the container.   The method for producing bubbles according to claim 1, wherein the step of injecting the solution into the container and the step of vibrating the container are performed so as to maintain a constant temperature of the solution.   The method for producing a bubble according to any one of claims 1 to 11, wherein the target substance includes an outline material constituting an outline of the bubble.   13. The outer shell material is an amphiphilic material containing at least one of proteins, polycationic lipids, phospholipids, higher fatty acids, sugars, sterols, surfactants, natural and synthetic polymers. The manufacturing method of the bubble of description.   14. The bubble production according to claim 13, wherein the outer shell is composed of a micelle composed of a monolayer of molecules of the amphiphilic material, or a liposome composed of a double layer of molecules of the amphiphilic material. Method.   The method for producing bubbles according to claim 1, wherein the target substance includes a drug.   The bubble production method according to any one of claims 1 to 15, wherein an average particle diameter of the bubbles is 10 nm to 1000 µm. Injecting a solution containing the target substance to a predetermined height of the container;
Vibrating the container at a rotational speed of 5000 rpm or more so that the solution repeatedly collides with the inner surface of the container in a state where the pressure in the container is greater than 1.0 atm,
The step of vibrating the container includes the step of vibrating the container by a reciprocating motion or a reciprocating motion and a rotating motion of the container in a horizontal direction,
The bubble manufacturing method, wherein the horizontal vibration width of the container is 0.3X to 0.8X (mm) when the height of the container is X (mm).

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