JP6539215B2 - Bubble manufacturing method - Google Patents

Description

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

  In recent years, the use of micron-sized (several hundreds of microns or less) or nano-sized (several hundreds of nanometers or less) bubbles has been considered in various fields such as medical care, food and fish farming, and wastewater treatment. In particular, in the medical field, there is known a method of performing ultrasound diagnosis on the chest or abdomen using microbubbles as an ultrasound contrast agent.

  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 diagnosis site, and imaging is performed by imaging the reflection wave (reflection echo) from the ultrasonic contrast agent. is there. And as an ultrasound contrast agent, the micro bubble (micro bubble) which consists of an outer shell comprised from protein, a lipid, etc., and the gas enclosed in the outer shell is used widely.

  Also, in recent years, an ultrasonic treatment method using this microbubble has been studied (for example, Patent Document 1). More specifically, microbubbles containing genes and drugs (drugs) are injected into the body and transported to the affected area through blood vessels. Then, when the microbubbles reach the vicinity of the affected area, ultrasonic waves are applied to the microbubbles to rupture the microbubbles. By doing so, the drug enclosed in the microbubble can be intensively administered to the affected area.

  As a method of 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 gas is dissolved under high pressure in a mixture containing a constituent material of the microbubble and physiological saline, and then the pressure is reduced to generate the microbubble in the mixture. In the gas-liquid two-phase flow swirling method, the mixture is stirred at high speed to generate a vortex of the mixture, and after sufficiently incorporating the gas in the vortex, the mixture is stopped by stopping the vortex. It is a method of generating micro bubbles in a liquid.

  In the method of producing microbubbles described above, it is necessary to prepare a mixture of at least 1 to 10 L in order to generate microbubbles. In addition, it was difficult to stably generate microbubbles with a small amount of mixed solution (for example, a mixed solution of several ml). In addition, there is a problem that there is a variation in the size of the microbubbles to be generated.

JP, 2002-209896, A

  The present invention has been made in view of the above-mentioned conventional problems, and an object thereof is to provide a method for producing a bubble capable of stably producing bubbles (micro bubbles or nano bubbles) of uniform size. . Another object of the present invention is to provide a container for bubble production used in a method for producing bubbles of uniform size.

Such an object is achieved by the present invention of the following (1) to (18).
(1) injecting a solution containing a 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 with the pressure in the container being greater than 1.0 atm. How to make a bubble.

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

  (3) The manufacturing method of the bubble as described in said (2) whose pressure of the said shock wave is 40 kPa-1 GPa.

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

  (5) The step of vibrating the container is performed to vibrate the container by reciprocating and / or rotational movement in the horizontal direction and / or vertical direction of the container (1) to (4). The manufacturing method of the bubble as described in any of.

  (6) The step of vibrating the container is performed to vibrate the container such that the instantaneous relative velocity 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 the above (1) to (5), which is

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

  (9) After the step of vibrating the container, after changing the pressure in the container, any one of the above (1) to (8) further including the step of re-oscillating the container at a rotational speed of 5000 rpm or more. The manufacturing method of the bubble as described in.

  (10) The method for producing a bubble according to (9), wherein the step of revibrating the container is performed such that the pressure is 1 to 10 atm higher than the pressure in the step of vibrating the container.

  (11) The method for producing a bubble according to (9) or (10), wherein the step of revibrating the container is performed at a rotation number different from the rotation number 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) in which the temperature of the solution is maintained constant. Bubble manufacturing method.

  (13) The method for producing a bubble according to any one of (1) to (12), wherein the target substance contains an outer shell material forming an outer shell of the bubble.

  (14) The outer shell material is an amphiphilic material containing at least one of a protein, a polycationic lipid, a phospholipid, a higher fatty acid, a saccharide, a sterol, a surfactant, and a natural or synthetic polymer. The manufacturing method of the bubble as described in (13).

  (15) The outer shell described in the above (14), which 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 method for producing a bubble according to any one of the above (1) to (15), wherein the target substance contains a drug.

  (17) The method for producing a bubble according to any one of the above (1) to (16), wherein the average particle diameter of the bubble is 10 nm to 1000 μm.

  (18) A container for bubble production used in the method for producing a bubble according to any one of (1) to (17) above.

  According to the present invention, it is possible to stably generate a large number of bubbles of uniform size in the solution simply by vibrating the container while pressurizing the inside of the container. As a result, it is possible to provide a container containing a large amount of bubbles of uniform size.

It is a perspective view which shows the state which cut | disconnected a part of bubble manufactured by the manufacturing method of the bubble of this invention. FIG. 1 (a) shows a state in which a part of the bubble in which the gas is enclosed in the shell is cut, and in FIGS. 1 (b) and 1 (c), the gas and the drug are enclosed in the shell. Shows a state in which a part of the bubble has been cut. It is a flowchart for demonstrating 1st Embodiment of the manufacturing method of the bubble of this invention. 3 (a) to 3 (d) are cross-sectional views for explaining the first embodiment of the method for producing a bubble of the present invention. In the process of vibrating the container shown in FIG.3 (c), it is the elements on larger scale for demonstrating the state which the liquid mixture and the inner surface (upper surface) of the container collided violently. It is a flowchart for demonstrating 3rd Embodiment of the manufacturing method of the bubble of this invention. It is a fragmentary sectional view showing the lid vicinity of the manufacture container used for a 4th embodiment of the manufacturing method of the bubble of the present invention. Fig.7 (a)-(f) is sectional drawing which shows typically the other structure of the container used for 5th Embodiment of the manufacturing method of the bubble of this invention. 8 (a) to 8 (c) are perspective views for explaining a sixth embodiment of the method for producing a bubble of the present invention. It is a figure for demonstrating the structure (handle is abbreviate | omitted) of the rubber plug vicinity of the mininate valve shown to Fig.8 (a). Fig.9 (a) is a top view of rubber stopper vicinity of a mininate valve, FIG.9 (b) is XX sectional drawing of Fig.9 (a). It is sectional drawing of the bubble containing container shown in FIG.8 (c). It is a perspective view for demonstrating the manufacture 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 manufacture container used for 9th Embodiment of the manufacturing method of the bubble of this invention. FIG. 13 (a) shows the manufacturing container in the disassembled state, and FIG. 13 (b) shows the manufacturing container in the 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. 14 (a) shows the manufacturing container in the disassembled state, and FIG. 14 (b) shows the manufacturing container in the assembled state. It is a figure for demonstrating the position of the opening part formed in the lid | cover of the manufacturing container shown in FIG.14 (b). Fig.15 (a) is a figure for demonstrating the state before puncturing the injection needle of a syringe in a rubber plug, and FIG.15 (b) is a bottom plate after clamping a needle from the rubber plug. It is a figure for demonstrating the state clamped to the part. FIG. 16 (a) is a graph showing the particle size distribution of the bubbles when the bubbles are produced at rotation speeds of 5000 rpm and 6500 rpm. FIG. 16B is a partially enlarged view of the graph shown in FIG. 16A in which the horizontal axis is in the range of 0 to 700 nm. FIG. 17 (a) is a graph showing the relationship between the number of revolutions of a closed vial and the average particle size of bubbles. FIG. 17 (b) is a graph showing the relationship between the number of revolutions of the closed vial and the content of bubbles. FIG. 18 (a) is a graph showing the relationship between the volume of gas sealed in a sealed vial and the average particle size of bubbles. FIG. 18 (b) is a graph showing the relationship between the volume of gas sealed in a sealed vial and the content of bubbles. It is a fluorescence-microscope image of the cerebrovascular pericyte culture | cultivation 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 cerebrovascular pericyte culture | cultivation 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 method for producing a bubble and a container for producing a bubble according to the present invention will be described based on preferred embodiments shown in the attached drawings.

  First, prior to the description of the method of producing a bubble of the present invention and the container for producing a bubble, the bubble produced by the method of producing a bubble of the present invention will be described.

  FIG. 1 is a perspective view showing a state in which a part of the bubble produced by the bubble producing method of the present invention is cut. Note that FIG. 1 (a) shows a state in which a part of the bubble in which the gas is enclosed in the shell is cut, and FIGS. 1 (b) and 1 (c) show the gas and the drug in the shell. The state which cut | disconnected a part of enclosed bubble is shown.

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

The shell 2 has a function of holding the gas 3 enclosed inside the bubble 1 in the bubble 1.
Such an outer shell 2 is composed of an amphiphilic material (shell material) having both hydrophobicity and hydrophilicity (substituents) in one molecule. Examples of amphiphilic materials include, but are not limited to, proteins such as albumin, polycationic lipids, phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phospholipids such as phosphatidyl ethanolamine, palmitic acid, stearic acid Or higher fatty acids, saccharides such as galactose, cholesterol, sterols such as sitosterol, surfactants, natural or synthetic polymers, fluorescent dyes, antibodies, labeled metals, etc. Two or more of them can be used in combination.

  Although not shown in FIG. 1, the amphiphilic material constituting the shell 2 is spherically arranged in the aqueous medium with the hydrophobic group on the inside and the hydrophilic group on the outside. Due to this property, the shell 2 is a micelle composed of a monolayer of molecules of amphiphilic material and a liposome composed of a bilayer of molecules of amphiphilic material.

  The gas 3 has a function to stabilize the shell 2 of the bubble 1. Such gas 3 is a gaseous substance at a temperature (about 20 ° C.) at the time of producing the bubble 1. Further, such bubble 1 is a gaseous substance also in the state where the bubble 1 is injected into the body, that is, at the temperature in the body (about 37 ° C.).

  Such gas 3 is not particularly limited, but, for example, air, nitrogen, oxygen, carbon dioxide, hydrogen, inert gas such as helium, argon, xenon, krypton, sulfur hexafluoride, sulfur difluoride Sulfur fluoride such as trifluoromethyl sulfur pentafluoride, methane, ethane, propane, butane, pentane, cyclopropane, cyclobutane, cyclobutane, cyclopentane, ethylene, propylene, propadiene, butene, acetylene, propyne, perfluoropropane, perfluoropropane And low molecular weight hydrocarbons such as fluorobutane and perfluoropentane or their halides, ethers such as dimethyl ether, ketones, esters and the like, and one or more of them may be used in combination It can be used. Among these substances, sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane are particularly preferable. These gas-enclosed bubbles 1 are highly stable in the body, and are more reliably transported through the blood vessels to the affected area or the diagnosis site.

  The particle diameter of the bubble 1 comprised with such a component changes by changing the conditions of each process of the manufacturing method of the bubble of this invention. That is, bubble 1 manufactured will have micron size (several hundreds of microns or less) or nano size (several hundreds of nanometers or less).

  Specifically, the average particle size (also referred to as the average particle size of the outer shell) 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 50 to 2000 nm More preferably, If the average particle size of the bubble 1 is within the above range, when the bubble 1 is injected into the body by intravenous injection, the particle diameter of the bubble 1 is sufficiently small, so the bubble 1 smoothly moves in the blood vessel by the blood flow. can do. In addition, bubbles of such a particle size are highly stable in blood vessels, and are reliably transported to the target site without disappearing while moving in blood vessels.

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

  Moreover, bubble 1 of the above-mentioned structure can be used also in fields other than the medical field, such as aquaculture of food and fish and shellfish, waste water treatment. In particular, when the average particle diameter of the bubble 1 is in the above range, the stability of the bubble 1 can be sufficiently high. The highly stable bubble 1 is easy to handle and can be applied to various fields.

Next, the bubble 1 shown in FIG. 1 (b) and FIG. 1 (c) will be described.
Bubble 1 shown in FIG.1 (b) and FIG.1 (c) can be manufactured by 2nd, 3rd, 10th embodiment of the manufacturing method of the bubble of this invention mentioned later. Such bubble 1 has an outer shell 2 constituting a shell of the bubble 1 and a gas 3 and a drug 4 enclosed in the outer shell 2. Such bubble 1 is used for ultrasound treatment and ultrasound diagnosis. 1 (b) shows the bubble 1 in which the drug 4 is enclosed in the outer shell 2 in the gaseous or solid state, and in FIG. 1 (c), the drug 4 is in the outer shell 2 in the liquid state. The bubble 1 enclosed is shown.

  The shell 2 has a function of holding the gas 3 and the drug 4 enclosed inside the bubble 1 in the bubble 1 and has a function of protecting the drug 4 until the bubble 1 is transported to the affected area. As a material which comprises the shell 2, the material similar to the shell 2 of the bubble 1 shown in FIG. 1 (a) mentioned above can be used.

  The gas 3 has a function to stabilize the shell 2 of the bubble 1. As the gas 3, the same substance as the gas 3 of the bubble 1 shown in FIG. 1A described above can be used.

  Moreover, in the bubble 1 shown in FIG. 1 (b) and FIG. 1 (c), the drug 4 is an effective component for treating various diseases such as prostate cancer, uterine fibroid, myocardial infarction, cerebral infarction and the like. Such a drug 4 is transported to the affected area in a state of being contained in the bubble 1, and is administered to the affected area by bursting the bubble 1 in the vicinity of the affected area by ultrasonic irradiation. The drug 4 is enclosed in the shell 2 together with the gas 3 or is contained or adsorbed in the shell 2 itself.

  Such a drug 4 is not particularly limited as long as it is effective for treatment of a disease, and includes genes, drugs and the like. Specifically, peptides, antibodies, oligosaccharides, polysaccharides, genes, oligonucleotides, antisense oligonucleotides, siRNAs, ribozymes, triple helix molecules, viral vectors, plasmids, small molecule organic compounds, anticancer agents, metals and the like can be mentioned. One or two or more of these can be used in combination.

  When the bubble 1 contains the drug 4, the content of the 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. It is further preferred that If the volume ratio of the drug 4 to the gas 3 is within the above range, the stability of the bubble 1 is improved, and the bubble 1 can be transported more reliably to the vicinity of the affected area. 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 area can be treated more efficiently.

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

  The particle diameter of bubble 1 composed of such components is changed by changing the conditions of each step of the method for producing bubble of the present invention, as in bubble 1 shown in FIG. 1 (a).

  In particular, if the average particle size of bubble 1 is 200 to 300 nm, it can be suitably used for cancer treatment. Specifically, in an affected area where cancer cells are present, there are new blood vessels that are smaller in diameter than normal blood vessels and extend from surrounding blood vessels to the cancer cells. If the average particle size of bubble 1 is within the above range, bubble 1 can be transported also into a neovascular having a small diameter. Therefore, the bubble 1 can be delivered directly to the cancer cells through the neovasculature. In addition, if the average particle size of the bubbles 1 is within the above range, some bubbles 1 can be transported directly to cancer cells by passing through the blood vessel wall. In addition, when 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 assured when an ultrasonic image is taken. It can be identified. Therefore, it can be suitably used for brain therapy (for example, intracerebral endovascular therapy etc.).

  In addition, the average particle diameter of bubble 1 shown to FIG. 1 (a)-(c) is observed by the laser diffraction and scattering method, a nanoparticle tracking analysis method, an electrical resistance method, AFM (Atomic Force Microscope), a laser microscope, for example. It can be measured by Moreover, as an apparatus for measuring AFM, for example, a resonance type particle measurement system (trade name: Archimedes) manufactured by Malvern can be used.

  The bubble 1 as described above can be produced by the method for producing a bubble of the present invention described below. Hereinafter, the method for producing a bubble of the present invention will be described in detail.

First Embodiment
1. Method of Producing Bubble Next, a first embodiment of the method of producing a bubble of the present invention will be described. The bubble 1 shown in FIG. 1 (a) described above can be manufactured by the bubble manufacturing method of the present embodiment.

  FIG. 2 is a flow chart for explaining the first embodiment of the method for producing a bubble of the present invention, and FIGS. 3 (a) to 3 (d) explain the first embodiment of the method for producing a bubble of the present invention FIG. 4 is a partial 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. 3C. 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.

  The manufacturing method of the bubble of this embodiment has five processes of process (S1)-process (S5), as shown in FIG. The step (S1) comprises a solution (mixed liquid) containing a target substance (the outer shell 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 the mixed solution to a predetermined height of the production container. The step (S3) is a step of filling the gas in the production container and sealing the production container in a state where the inside of the production container is pressurized. The step (S4) is a step of vibrating the production container at a predetermined number of rotations so that the mixed solution repeatedly collides with the inner surface of the container. Step (S5) is a step of leaving the production container stationary. These steps will be sequentially described below.

[S1] Preparation Step First, the material (shell material) constituting shell 2 of bubble 1 and the aqueous medium are placed in a mixed liquid preparation container (hereinafter simply referred to as "preparation container") to dissolve the shell material in the aqueous medium The mixture 10 is prepared. That is, after adding a predetermined amount of shell material and an aqueous medium in a preparation container, it stirs and dissolves shell material in an aqueous medium. The order of placing the shell material and the aqueous medium in the preparation container is not particularly limited. As a method of dissolving the shell material in an aqueous medium, for example, stirring with a stirrer, ultrasonication, or the like can be used.
The amphiphilic material described above is used as the shell material.

  The content of the shell material in the liquid mixture 10 is not particularly limited as long as bubbles 1 are formed in the liquid mixture 10 in the step (S4). Although the preferable content of the shell material varies depending on the combination of each type of shell material and aqueous medium, it is preferable that the shell material is contained in the mixture 10 at a concentration above the critical micelle concentration (CMC). Specifically, the content of the shell material contained in the liquid mixture 10 is preferably 0.01 to 50 wt%, and more preferably 0.1 to 20 wt%.

  As a result, the concentration of the shell material in the liquid mixture 10 is more reliably equal to or higher than the critical micelle concentration, and therefore, the shell 2 (liposomes, micelles) can be formed in the liquid mixture 10 with certainty. Therefore, in the step (S4) to be described later, the gas 3 can be easily taken into the liposome or micelle, and the bubble 1 having a desired particle diameter can be easily generated in the mixture liquid 10. In addition, the stability of the shell 2 is enhanced, and the bubble 1 can be more reliably prevented from being unintentionally ruptured. Furthermore, variation in the size of the generated bubble 1 can be reduced. That is, bubbles 1 of uniform size can be generated.

  The aqueous medium is not particularly limited, and, for example, water (distilled water), physiological saline (phosphate buffered saline), 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 mixed solution 10 to be prepared is preferably 50 to 99.99 wt%, and more preferably 80 to 99.0 wt%. Thereby, the shell material can be sufficiently dissolved in the aqueous medium, and a more uniform mixture 10 can be obtained.

Next, the production container 20 (the bubble production container of the present embodiment) is prepared.
The manufacturing container 20 has a container body 21 for containing the liquid mixture 10 and a lid 22 for sealing the container body 21.

  Although the container body 21 is not particularly limited, it is preferable that the outer shape as shown in FIG. 3A has a cylindrical shape. In the present embodiment, a vial having a volume 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 a small vial having such a capacity is used as the container body 21, the sealed space in the container body 21 when the container body 21 is sealed by the lid 22. Since an appropriate pressure is applied to the mixture 10, bubbles of uniform size can be stably obtained. In particular, in the case of a vial having a volume of about 0.5 to 1.5 ml, a bubble-containing mixed liquid of about 0.3 to 0.6 ml which is required for one ultrasonic diagnosis in one production container 20 Can be manufactured. In this case, since the bubble-containing mixed solution in one manufacturing container 20 can be used up at the time of ultrasonic diagnosis, it is possible to eliminate the waste of the manufactured bubble-containing mixed solution.

  Thus, the vial bottle (volume: about 0.5 to 20 ml) having a small volume 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 disk-shaped rubber plug (septum) 221 in close contact with the bottle opening of the container body 21 and the rubber plug 221 to the bottle opening of the container body 21. And a fastening portion 222.

  Although the rubber plug 221 is not particularly limited, for example, a rubber plug made of silicon can be used.

The tightening portion 222 is configured to cover the edge of the rubber plug 221. Further, on the inner peripheral surface on the bottle opening side of the tightening portion 222 and on the outer peripheral surface on the bottle opening side of the container main body 21, screw grooves formed so as to be mutually screwable are formed respectively (not shown) By screwing them together, the rubber plug 221 is fixed in a state of being in close contact with the mouth of the container body 21. In addition, by caulking the tightening portion 222 to the bottle opening of the container main body 21, the container main body 21 and the tightening portion 222 can be fixed with the rubber plug 221 in close contact with the bottle opening of the container main body .
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, a container having a relatively large capacity is used as the preparation container, while the production container 20 (the container main body 21) has a relatively small capacity. A container can be used. In this case, by preparing a large amount of the mixed solution 10 having a uniform composition in the preparation container and dividing the mixed solution 10 into a plurality of manufacturing containers 20, the size of the bubbles generated in each manufacturing container 20 ( Particle size), the amount can be made uniform.

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 Mixed Liquid into Manufacturing Container The prepared mixed liquid 10 is injected to a predetermined height of the container body 21 (manufacturing container 20). In this embodiment, as shown to Fig.3 (a), it injects to Y [mm]. Therefore, as shown to Fig.3 (a), the container main body 21 of the state into which the liquid mixture 10 was inject | poured has the cavity 11 in the upper part.

  In the present embodiment, the height of the container body 21 is X [mm] in a state where the container body 21 (production container 20) into which the liquid mixture 10 has been injected is allowed to stand horizontally, and the liquid mixture 10 in the container body 21 is When the height of the liquid surface is Y [mm], it is preferable to satisfy the relationship of 0.2 ≦ Y / X ≦ 0.7. By satisfying the above relationship, there is a gap 11 of a sufficient size, so in the step (S4), the mixed solution 10 is vigorously collided with the upper and lower surfaces and the side surfaces (especially upper and lower surfaces) of the manufacturing container 20. be able to. Due to this collision, a shock wave is generated in the mixture 10, and the bubble 1 can be easily formed in the mixture 10.

  It is more preferable that the relationship satisfies 0.3 ≦ Y / X ≦ 0.5, and still more preferably 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 Manufacturing Container Next, the container body 21 is filled with the gas 3, and the inside of the manufacturing container 20 is sealed in a pressurized state (see FIG. 3B). Specifically, after the void portion 11 of the container body 21 into which the liquid mixture 10 has been injected is purged with the gas 3, the lid 22 is inserted into the opening (bottle neck) of the container body 21. Thereby, the mixture 10 and the gas 3 are sealed in the production container 20.

Specifically, a syringe filled with gas 3 is prepared. Then, the injection needle of the syringe is punctured into the rubber plug 221. Thereafter, additional gas 3 is added from the syringe into the manufacturing container 20. Thereby, the inside of the manufacturing container 20 is pressurized. Thereafter, by removing the injection needle from the lid 22 (rubber plug 221), it is possible to obtain the manufacturing container 20 sealed in a state where the inside of the manufacturing container 20 is pressurized by the gas 3.
The various gases described above are used as the gas 3.

  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 void 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, more preferably 2 to 5 atm. Thereby, a part of the gas 3 dissolves in the mixture 10.

  By dissolving the gas 3 in the mixture 10, the gas 3 in the mixture 10 is taken into the bubble 1 generated in the step (S4). Thereby, a large amount of bubbles 1 can be generated in the mixture 10 in the step (S4).

  Further, by setting the pressure in the production container 20 to an arbitrary value larger than 1.0 atm, the particle size and content of the bubbles 1 generated in the mixed liquid 10 can be adjusted.

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

  At this process, the manufacturing container 20 (lower figure of FIG.3 (c)) sealed at process (S3) is vibrated upward (figure of the middle of FIG.3 (c)). Thereby, the liquid mixture 10 moves to the vicinity of the middle of the production container 20. Further, when the manufacturing container 20 is 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 of FIG. 3C). At this time, as shown in FIG. 4, a shock wave is generated. The pressure of this shock wave makes the shell material in the mixture 10 a bubble 1. The bubble 1 contains a gas 3 dissolved in the mixture 10 and a gas 3 dissolved or mixed in the mixture 10 by vibration.

  On the other hand, the manufacturing container 20 (the upper view of FIG. 3C) is vibrated downward (the middle view of FIG. 3C). Thereby, the liquid mixture 10 moves to the vicinity of the middle of the production container 20. Further, when the manufacturing container 20 is vibrated downward, the mixed solution 10 moves to the lower part of the manufacturing container 20 and collides with the lower surface of the manufacturing container 20 (lower part of FIG. 3C). Also at this time, as shown in FIG. 4, a shock wave is generated.

  Further, when vibrating the manufacturing container 20 in the vertical direction, the mixed solution 10 also collides with the inner side surface of the manufacturing container 20. Also at this time, as shown in FIG. 4, a shock wave is generated.

  By repeatedly performing the above-described operation, it is possible to stably generate a large amount of bubbles 1 of uniform size in the liquid mixture 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 bubbles 1 of sufficiently fine and uniform size. As a result, the size (pressure) of the shock wave generated when the liquid mixture 10 collides with the production container 20 becomes sufficiently large, and the bubbles 1 generated in the liquid mixture 10 are refined to make the size uniform. be able to. In addition, when the rotational speed of the manufacturing container 20 is set to a relatively low value within the above range, the size 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 number of rotations is set to be high, the size of the generated shock wave becomes large, so it is possible to generate bubbles 1 with a relatively small particle size.

  The rotation speed of the production container 20 is 5000 rpm or more, preferably 5500 rpm or more, and more preferably 6000 to 20000 rpm. By setting the rotation speed of the manufacturing container 20 within the above range, it is possible to more reliably prevent the bubbles 1 generated by the vibration from collapsing due to a collision or coalescing and coarsening. As a result, it is possible to generate a large amount of bubbles 1 of more uniform size in the mixed liquid 10 while reducing the particle size of the bubbles 1.

  As a device capable of vibrating 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 (Beltins) may be used.

  Moreover, it is preferable that the pressure of the shock wave which generate | occur | produces when the liquid mixture 10 and the manufacturing container 20 collide will be 40 kPa-1 GPa. When the pressure of the shock wave generated at the time of the collision between the mixture 10 and the production container 20 falls within the above range, the bubbles 1 generated in the mixture 10 can be further miniaturized 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 liquid mixture 10 and the production container 20 increases, finer bubbles 1 can be generated.

  When vibrating the production container 20, the vibration width of the production container 20 in the vertical direction is preferably about 0.7 X to 1.5 X [mm], and about 0.8 X to 1 X [mm]. More preferable. Thereby, when the manufacturing container 20 vibrates, the mixed solution 10 can be reliably collided with the lower surface of the manufacturing container 20 and the lid 22, and the number of collisions between the mixed solution 10 and the lower surface of the manufacturing container 20 and the lid 22 is sufficient. You can do more. Also, by vibrating the production container 20 with a sufficient vibration width in this manner, 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 becomes sufficiently large. As a result, a large amount of fine bubbles 1 can be generated in the mixture 10.

  Further, when reciprocating the manufacturing container 20 in the vertical direction, it is preferable to vibrate the manufacturing container 20 in the horizontal direction as well. As a result, the mixed solution 10 also collides with the side surface inside the manufacturing container 20, so that more vibration waves can be generated in the mixed solution 10. The vibration width in the horizontal direction of the production container 20 is preferably about 0.3X to 0.8X [mm], and more preferably about 0.5X to 0.7 X [mm]. Thereby, the above-mentioned effect becomes more remarkable.

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

  In addition, it is preferable that it is about 10 to 120 second, and, as for the time which vibrates the manufacturing container 20 on the said conditions, it is more preferable that it is about 30 to 60 second. By setting the vibration time of the production container 20 within the above range, the number of times the mixed solution 10 collides with the production container 20 is sufficiently increased, so a large amount of bubbles 1 can be generated in the mixed solution 10. In addition, the amount of bubbles 1 generated in the liquid mixture 10 can be further increased by setting the vibration time of the manufacturing container 20 to be long within the above range.

  The particle diameter of the bubble 1 generated in the liquid mixture 10 can be adjusted by changing the pressure of the gas 3 in the manufacturing container 20 and the number of rotations of the manufacturing container 20 within the above-described range. 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 with the pressure in the production container 20 being 1.0 to 2 atm. In addition, nano bubbles of several tens to several hundreds of nanometers in size 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 manufacturing container 20 may be vibrated such that the manufacturing container 20 rotationally moves in the horizontal direction and / or the vertical direction. Even in this case, the mixed solution 10 in the manufacturing container 20 repeatedly collides with the upper and lower surfaces and the side surfaces of the manufacturing container 20 to generate a shock wave. Even with such a vibration method, a large amount of bubbles 1 of uniform size can be stably generated in the mixed solution 10.

[S5] Step of leaving production container stationary After making the production container 20 vibrate under the above conditions, the production container 20 is left to stand (see FIG. 3D). As a result, a large amount of bubbles 1 (see FIG. 1A) of uniform size can be stably manufactured in the manufacturing container 20. At the same time, a production container 20 containing a large amount of uniformly sized bubbles 1 is obtained.

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

  Through the above steps (S1) to (S5), bubbles 1 having an average particle diameter of about 10 nm to about 1000 μm are produced.

  In addition, in the manufacturing method of the conventional bubble, the large-scale reflux apparatus and the various systems (a tube, a nozzle, a compressor, etc.) which comprise the manufacturing apparatus of a bubble were required. Therefore, it has been difficult to maintain a clean and aseptic environment when producing bubbles used in the food field, medical field and the like. On the other hand, in the present invention, since the production container 20 for producing the bubble is sufficiently small, no large-scale system is required. Furthermore, according to the present invention, the bubble 1 can be produced in a closed, clean and aseptic environment (the sealed production container 20), so that it can be suitably used in the food field, medical field and the like.

  The bubble 1 obtained as described above can be stably present in the mixed solution 10. Therefore, the obtained bubble-containing container can be stored at room temperature for a long time. Specifically, it can be stored over a period of up to 6 to 24 months. In addition, even after storage for such a long period of time, the stability of the bubble 1 in the mixed solution 10 is high, and therefore, it is possible to use it without the need 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 held down. Therefore, the bubble-containing container obtained as described above has an advantage of being easy to handle for medical institutions and the like.

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 plug 221 of the lid 22. Next, the bubble-containing mixed solution is aspirated from the bubble-containing container. Then, the injection needle is removed from the rubber plug 221 and injected into the body from the patient's vein or the like. Thereafter, the bubble 1 is carried to the affected area by the blood flow.

  At the time of ultrasound diagnosis, an ultrasound signal of frequency and output adjusted so as not to burst the bubble 1 is sent to the bubble 1 in anticipation of the timing when the bubble 1 reaches the site to be diagnosed. Thereafter, a signal (reflection echo) reflected from the bubble 1 is received and data processed to display an image of a blood vessel. Thereby, ultrasonic diagnosis can be performed.

  In addition, as a device which receives irradiation of an ultrasonic wave and the reflected wave from the bubble 1, a well-known ultrasonic probe can be used.

  The bubble-containing container obtained as described above can be applied to various fields other than ultrasonic diagnostic applications. For example, the bubble 1 in the bubble-containing container obtained as described above has a bactericidal effect on water and food, and also has an effect of maintaining the freshness of the food. Furthermore, in the liquid containing bubble 1, water, and oil (hydrophobic component), a large amount of oil can be mixed with water. It is also possible to suppress and separate | separate with the water | moisture content in a foodstuff, and an oil component, and to cook using this effect. Therefore, it is also possible to use the obtained bubble-containing mixture in the food field.

  Further, in the above description, by performing the steps (S1) to (S5), a large number of bubbles 1 (see FIG. 1 (a)) of uniform size can be stably produced in the production container 20 in a stable manner. it can. However, the manufacturing method of the bubble of this embodiment is not limited to this. For example, after the step (S5), the step (S4) and the step (S5) may be repeated at least once or more. By repeatedly performing step (S4) and step (S5), bubble 1 of 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. 1 (b) and FIG. 1 (c) described above can be manufactured by the bubble manufacturing method of the present embodiment.

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

1. Method of Producing Bubble In the present embodiment, the target substance contains the shell material and the drug 4. That is, in the bubble manufacturing method of the present embodiment, except that the mixed solution 10 prepared in the step (S1) described above contains the drug 4 in addition to the shell material and the aqueous medium, the method of the first embodiment described above It is the same as the bubble manufacturing method.

[S1] Preparation Step In this embodiment, the material constituting the shell 2 of the bubble 1 (shell material), the drug 4 and the aqueous medium are placed in a mixed liquid preparation container (hereinafter simply referred to as "preparation container") to form the shell material. And drug 4 are dissolved in an aqueous medium to prepare mixture 10. That is, after the shell material, the drug 4 and the aqueous medium are added in predetermined amounts into the preparation container, the shell material and the drug 4 are dissolved in the aqueous medium by stirring. The order of placing the shell material, the drug 4 and the aqueous medium in the preparation container is not particularly limited. As a method of dissolving the shell material and the drug 4 in an aqueous medium, for example, stirring with a stirrer, ultrasonic treatment, or the like can be used.
The types and contents of the shell material and the aqueous medium are the same as in the first embodiment described above.

  As the drug 4, the aforementioned gene, drug or the like is used. The content of the drug 4 contained in the mixed solution 10 to be prepared is preferably 0.1 to 50 wt%, and more preferably 20 to 50 wt%. This allows the produced bubble 1 to contain a sufficient amount of the drug 4. As a result, it is possible to produce the bubble 1 with a better therapeutic effect on the affected area. When the drug 4 constitutes the shell 2 instead of the shell material, the mixture 10 may not contain the shell material.

  By performing the steps (S2) to (S5) in the same manner as in the first embodiment described above using the liquid mixture 10 prepared in this manner, bubbles 1 of uniform size in the production container 20 ( A large amount of FIG. 1 (b) can be stably manufactured. At the same time, a production container 20 containing a large amount of uniformly sized bubbles 1 is obtained.

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

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

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

  In the case of ultrasonic treatment, when the bubble 1 reaches the vicinity of the affected area, the ultrasonic wave is irradiated to burst the bubble 1. Thus, the drug 4 in the bubble 1 can be intensively administered to the affected area.

  In addition, such a bubble-containing mixed solution is also effective in performing ultrasonic treatment and ultrasonic diagnosis in combination. Specifically, ultrasonic waves of such a frequency and intensity that the bubble 1 does not burst are irradiated to the bubble 1 in the blood vessel, and the reflected wave is monitored. Thereby, the position of bubble 1 can be grasped correctly. When the bubble 1 reaches the target affected area, the bubble is ruptured by ultrasonic wave. Thereby, 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 to the aqueous medium mentioned above as gas 3, when predetermined time passes after manufacturing bubble 1, gas 3 in outer shell 2 is, Dissolve in aqueous medium. Thereafter, when the gas 3 in the shell 2 completely dissolves in the aqueous medium, only the drug 4 is enclosed in the shell 2. That is, in such a case, the bubble 1 is a liposome or micelle in which only the drug 4 is encapsulated in the shell 2. Liposomes or micelles thus obtained can also be used as a drug.

In addition, it is preferable that it is about 0.1-30 W / cm < ² > about the output of the ultrasonic wave for making the bubble 1 inject | poured in the body burst at the time of an ultrasonic treatment, about 0.5-10 W / cm < ² > Is more preferred. By setting the output of the ultrasonic wave within the above range, the bubble 1 can be more reliably ruptured, and the damage to the normal cells around the affected area can be reliably prevented. Moreover, when the output of an ultrasonic wave is in the said range, it is preferable that it is about 10 to 120 second, and, as for the irradiation time of an ultrasonic wave, it is more preferable that it is about 30 to 60 second.

  Further, the frequency of the ultrasonic wave to be irradiated in 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, it is possible to burst the bubble with lower ultrasonic power.

  The bubble producing method of the second embodiment also produces the same action and effect as the bubble producing method of the first embodiment.

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 method for producing a bubble of the present invention.

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

  The bubble manufacturing method of the present embodiment includes, as shown in FIG. 5, steps (S6) and (S7) after 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 at which the manufacturing container is vibrated.

  In the bubble manufacturing method of the present embodiment, when the rotation number is not changed in step (S7) (in FIG. 5, “NO” is selected in step (S7)), and further, step (S4 ′) and step It has (S5 '). On the other hand, in the step (S7), when changing the number of rotations (in FIG. 5, "YES" is selected in the step (S7)), the step (S8) and the step (S9) are further included. Furthermore, the manufacturing method of the bubble of this embodiment has the process (S10) which changes the pressure in a manufacture container. Hereinafter, each process after process (S6) is demonstrated one by one.

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

[S6] Step of Changing Pressure in Manufacturing Container The pressure in the bubble-containing container (the manufacturing container 20) after the step (S5) of the first embodiment described above is changed.

(1) In the case where the pressure in the production container is made higher than the pressure in the step (S3) The inside of the production container 20 is pressurized in the same manner as the step (S3) described above. The gas 3 to be injected may be the same as or different from the gas 3 used in the step (S3), but from the viewpoint of the stability of the bubble finally generated, the same gas 3 is used. preferable.

  In such a manufacturing container 20, since the inside of the manufacturing container 20 is more pressurized than the pressure in the manufacturing container 20 in the step (S3), the amount of the gas 3 dissolved in the mixed liquid 10 is the step (S3). The amount of the gas 3 dissolved in the mixture 10 in Therefore, when the production container 20 is vibrated again in the step (S4 ') or the step (S8) described later, the gas 3 in the mixed solution 10 is easily taken into the generated bubble 1, and as a result, the bubble 1 is produced. The amount of generation of In addition, a pressure greater than the pressure applied to the bubble 1 generated in step (S4) is applied to the mixture 10. As a result, since the bubble 1 in the production process is compressed at a higher pressure, the particle size of the bubble 1 tends to be smaller. Therefore, bubble 1 of smaller particle size can be generated than bubble 1 generated in step (S4).

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

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

  In such a production container 20, when the production container 20 is vibrated again in step (S4 ') or step (S8) described later, the pressure applied to the liquid mixture 10 is the mixture generated in the step (S4). It is less than the pressure applied to solution 10. As a result, the degree to which the bubble 1 in the formation process is compressed becomes smaller, and the particle size of the bubble 1 tends to be larger. Therefore, bubble 1 of larger particle size can be generated than bubble 1 generated in step (S4).

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

[S7] Step of Setting Number of Rotations for Vibrating Manufacturing Container After changing the pressure in the manufacturing container 20 as described above, the number of rotations for re-oscillating the container is set.

  The number of rotations at the time of revibrating the container is set to 5000 rpm or more, as in the step (S4) described above.

  In the step (S7), when the number of revolutions at the time of revibrating the container is not changed from the number of revolutions in the step (S4), that is, when "NO" is selected in the step (S7) in FIG. Perform (S4 ').

  On the other hand, in the step (S7), when changing the number of rotations at the time of revibrating the container from the number of rotations in the step (S4), that is, when “YES” is selected in the step (S7) in FIG. The step (S8) is performed.

[S4 ′] Step of Revibrating the Production Container As described above, the production container 20 with the pressure changed is revibrated at the same rotational speed as the above-described step (S4). Thereby, bubbles 1 having a particle diameter different from that of the bubbles 1 generated in the step (S4) are generated in the liquid mixture 10.

  In this step, the particle diameter of bubble 1 newly generated in the present embodiment changes in accordance with the pressure changed in step (S6) in order to re-oscillate the production container 20 at the same rotation speed as step (S4). Do. That is, since the particle diameter of bubble 1 can be adjusted by changing the pressure, bubble 1 having 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, it can manufacture the bubble 1 which has a different particle size more simply.

[S5 '] Step of leaving production container stationary After making the production container 20 vibrate under the above conditions, the production container 20 is left to stand in the same manner as step (S5). Thereby, it is possible to stably produce a large amount of bubbles 1 of different sizes in the production container 20. At the same time, a production container 20 (bubble-containing container) containing a large amount of such bubbles 1 is obtained.
After standing still, a process (S10) is performed.

[S8] Step of Revibrating the Manufacturing Container The manufacturing container 20, in which the pressure is changed, is re-oscillated at a rotational speed different from that of the step (S4) described above. Thereby, bubbles 1 having a particle diameter different from that of the bubbles 1 generated in the step (S4) are generated in the liquid mixture 10.

(1) In the case of revibrating the production container at a rotational speed higher than that of the step (S4) The above-described step (S4) except that the rotational frequency at which the production container 20 is vibrated is made higher than the rotational number in the step (S4). In the same manner as in the above, the production container 20 is vibrated again.

  In this case, the number of revolutions of the production container 20 is not particularly limited as long as it is higher than the number of revolutions in the step (S4), but is preferably 6,000 to 20,000 rpm and more preferably 7,000 to 20,000 rpm. Thereby, since the manufacturing container 20 is vibrated at a rotation number larger than the rotation number in the step (S4), it is possible to generate the bubble 1 having a particle diameter smaller than that of the bubble 1 generated in the step (S4). In addition, by setting the rotation number of the production container 20 within the above range, it is more reliably prevented that the bubbles 1 generated in the step (S4) and the present step collapse due to collision or coalescence. can do. Thereby, the bubble 1 produced | generated in process (S4) and the bubble 1 of the particle size smaller than the bubble 1 produced | generated in process (S4) can be manufactured.

(2) When the production container is re-vibrated at a rotation speed lower than that of the step (S4) The above-described step (S4) is performed except that the rotation number at which the production container 20 is vibrated is lower than the rotation number in the step (S4). In the same manner as in the above, the production container 20 is vibrated again.

  In this case, the number of rotations of the production container 20 is not particularly limited as long as it is lower than the number of rotations in the step (S4), but is preferably 5000 to 9000 rpm, and more preferably 5500 to 7500 rpm. Thereby, since the manufacturing container 20 is vibrated at a rotation speed lower than the rotation 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). In addition, by setting the rotation number of the production container 20 within the above range, it is more reliably prevented that the bubbles 1 generated in the step (S4) and the present step collapse due to collision or coalescence. can do. Thereby, the bubble 1 produced | generated in process (S4) and the bubble 1 of the particle size larger than the bubble 1 produced | generated in process (S4) can be manufactured.

  In this step, the particle diameter of the bubble 1 newly generated in the present embodiment changes the pressure in the production container 20 and causes re-vibration in order to re-vibrate the production container 20 at a rotation speed different from that of the step (S4). It changes with the number of rotations. As described above, by changing both the pressure in the manufacturing container 20 and the number of revolutions at the time of re-oscillation, it is possible to manufacture the bubble 1 having a particle diameter largely different from the particle diameter obtained in the first embodiment described above. it can. Therefore, this process is advantageous when producing bubbles 1 having large differences in average particle size.

[S9] Step of leaving production container stationary After making the production container 20 vibrate in step (S8), the production container 20 is left to stand in the same manner as step (S5). Thereby, bubbles 1 of different sizes 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.
After standing still, a process (S10) is performed.

[S10] The process of changing the pressure in the manufacturing container again If the pressure in the manufacturing container 20 is not changed, that is, if "NO" is selected in the step (S10) in Fig. 5, the manufacturing of the bubble of this embodiment The method ends. Thereby, the 1st bubble 1 and the 2nd bubble 1 which have an average particle diameter which is respectively different in an average particle diameter in the range of 10 nm-1000 micrometers are manufactured.

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

  In addition, when changing a pressure repeatedly in a process (S10), the bubble 1 which has a mutually different several average particle diameter of the number according to the frequency | count which changes a pressure can be manufactured.

  The bubble-containing container obtained as described above contains bubbles 1 having different particle sizes in the liquid mixture 10. The size difference makes the passage of bubble 1 easier in the blood vessel, and the site to be transported also changes (for example, the smaller the size of bubble 1, the more it can be transported to the tip of the capillary). The bubble-containing mixture thus obtained can be used multi-facetedly for the purpose of ultrasonic treatment.

  The bubble producing method of the third embodiment produces the same action and effect as the bubble producing 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 manufacturing method of the bubble of the fourth embodiment will be described focusing on the difference from the manufacturing method of the bubble of the first to third embodiments, and the description of the same matters will be omitted.

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

As a constituent material of such a bottom plate portion 223, for example, various ceramic materials and various metal materials can be mentioned, but a material having a density (2000 kg / m ³ or more) of glass or more is preferable. As such materials, stainless steel such as cast iron (density: about 7000 to 7700 kg / m ³ ), chromium nickel steel 18/8 (density: about 7900 kg / m ³ ), V2A steel (density: about 7900 kg / m ³ ) Steel, aluminum (density: about 2700 kg / m ³ ), germalmin (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 them may be used in combination Can be used. Among these materials, it is particularly preferable to use iron or an iron alloy such as stainless steel from the viewpoint of high specific gravity and high corrosion resistance to the components of the liquid mixture 10.

  The mass of the bottom plate portion 223 made of such a material is larger than that of a member formed of a material having a small specific gravity (density) such as synthetic rubber or resin. By increasing the mass of the bottom plate portion 223, it is possible to further increase the size of the shock wave generated when the liquid mixture 10 collides with the upper surface (bottom plate portion 223) of the manufacturing container 20 in step (S4). As a result, fine bubbles 1 can be more easily and stably generated in the liquid mixture 10.

  When such a lid 22 is used, the rubber plug 221 is punctured so that the injection needle of the syringe passes through the opening 224 of the bottom plate 223 in step (S3). Then, as in the above-described embodiment, the inside of the manufacturing container 20 is pressurized by further adding the gas 3 from the syringe into the manufacturing container 20. Thereafter, by removing the injection needle from the lid 22 (rubber plug 221), it is possible to obtain the manufacturing container 20 sealed in a state where the inside of the manufacturing container 20 is pressurized by the gas 3.

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

  The bubble producing method of the fourth embodiment produces the same operation and effect as the bubble producing 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.

  Fig.7 (a)-(f) is sectional drawing which shows typically the other structure of the container used for 5th Embodiment of the manufacturing method of the bubble of this 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 manufacturing method of the bubble of the fifth embodiment will be described focusing on the difference from the manufacturing method of the bubble of the first to fourth embodiments, and the description of the same matters will be omitted.

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

  In the present embodiment, the production container 20 (the container for producing a bubble according to the present embodiment) has the container main body 21 of various shapes shown in FIGS. 7A to 7D and the upper surface of each container main body 21. And a lid (not shown). As shown in FIGS. 7 (a) to 7 (d), the inner surface of the container body 21 includes at least one of a concave surface, a convex surface and an uneven surface.

  The container body 21 shown in FIG. 7A is a container in which the upper surface and the lower surface are convex surfaces projecting inward. The manufacturing container 20 shown in FIG. 7B is a container in which the upper surface and the lower surface are uneven surfaces. The manufacturing container 20 shown in FIG. 7C is a container in which the upper surface and the lower surface are concave surfaces protruding outward. The manufacturing container shown in FIG. 7 (d) is a container whose side is a convex surface curved toward the inside of the manufacturing container 20.

  Such a surface (concave, convex or irregular surface) has a large surface area as compared to a flat surface. Therefore, in the step (S4) described above, the area in which the liquid mixture 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 the pressure of the generated shock wave differs depending on the shape. As a result, it is possible to generate a large amount of bubbles 1 of different particle sizes in the liquid mixture 10.

  Moreover, the height of the container main body 21 can be made higher than the container main body 21 of 1st-4th embodiment mentioned above, as shown in FIG.7 (e). 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 liquid mixture 10 becomes long. Therefore, the magnitude of the shock wave generated at the time of the collision can be increased. Thereby, the size of bubble 1 manufactured in mixed solution 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 1st-4th embodiment mentioned above, as shown in FIG.7 (f). When such a container body 21 is used, when the manufacturing container is vibrated in the steps (S4, S4 'and S8), the moving distance of the liquid mixture 10 becomes short. Therefore, since the number of times the mixed solution 10 collides with the inner surface of the manufacturing container can be increased, it is possible to apply more pressure to the mixed solution 10 by the shock wave. Thereby, more bubbles 1 can be produced in the mixture 10.

  The bubble producing method of the fifth embodiment produces the same action and effect as the bubble producing method of the first to fourth embodiments.

Sixth Embodiment
Next, a sixth embodiment of the method for producing a bubble of the present invention will be described.

  8 (a) to 8 (c) are perspective views for explaining a sixth embodiment of the method for producing a bubble of the present invention. FIG. 9 is a view for explaining the configuration (handle is omitted) in the vicinity of the rubber plug of the mininate valve shown in FIG. 8 (a), and FIG. 9 (a) is an upper surface near the rubber plug of the mininate valve. It is a figure, FIG.9 (b) is XX sectional drawing of Fig.9 (a). FIG. 10 is a cross-sectional view of the bubble-containing container shown in FIG. 8 (c).

  In the following description, the upper side in FIGS. 8 (a) to (c), 9 (b), 10, and 10 in FIG. 9 (a) is referred to as "upper". The lower side in FIG. 9 (b), FIG. 9 (b), FIG. 10 and the paper surface back side in FIG. 9 (a) are called "down". Moreover, the left side in FIG. 8 (a)-(c) is called "left", and the right side in FIG. 8 (a)-(c) is called "right."

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

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

[S1] Preparation Step A production container 20 (a container for bubble production of the present embodiment) as shown in FIG. 8A is prepared.

  In the present embodiment, the manufacturing container 20 has the container main body 21 and the lid 22 similarly to the manufacturing container 20 of the first embodiment described above. As shown in FIG. 8A, the lid 22 is provided on the upper side thereof with a mininate valve 30, and a tube 33 connecting the rubber plug 221 and the mininate valve 30.

  The mininate valve 30 has a valve main body 31 having a through hole 311, a rubber plug 32 embedded in the through hole 311 and having a pipe line 321 penetrating in the thickness direction (vertical direction), opening and closing the pipe line 321 And a handle 34 for controlling the

  The valve body 31 has a substantially rectangular parallelepiped shape, and a through hole 311 penetrating in the thickness direction (vertical direction) is formed in the vicinity of 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, silicone 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. A conduit 321 in which the injection needle 41 of the syringe 40 is insertable is formed substantially at the center of the rubber plug 32. Further, the rubber plug 32 is formed with a through hole 322 which is orthogonal to the pipe line 321 and penetrates in the width direction (left and 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 main body 31 and the respective knobs 341, and the through holes 322 of the rubber plug 32 and the through holes 312 of the valve main body 31 And a shaft 342 slidably inserted. A through hole 343 is formed in a part of the shaft 342 in the thickness direction (vertical direction in FIG. 8A).

  Although the tube 33 is not particularly limited, it is made of, for example, a tube made of silicon. The upper end portion of the tube 33 is in communication with the conduit 321 of the rubber plug 32, and the lower end portion of the tube 33 is in communication with the internal space (void 11) of the container main body 21 via the rubber plug 221. . In other words, the conduit 321 and the internal space of the container body 21 are in communication 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 into contact with the left end of the valve main body 31. The through hole 343 of the shaft 342 and the conduit 321 overlap (communicate) in a plan view (top view). As a result, the conduit 321 is opened, and the conduit 321, the tube 33, and the internal space of the container main body 21 communicate with each other (see FIGS. 8A and 9B). On the other hand, as shown in FIG. 8B, the knob 341 on the right side is pushed to the left, and the knob 341 on the right side is brought into contact with the end on the right side of the valve body 31 to make a plan view (top view) The position of the through hole 343 of the shaft 342 deviates from the position of the conduit 321 at the point of. As a result, the conduit 321 is closed, and the inside of the manufacturing container 20 is sealed.

  Further, in the present embodiment, the lid 22 includes a rubber plug 221, a tightening portion 222, and a bottom plate portion 223 as in the lid 22 shown in FIG. 6 (see FIG. 10). As shown in FIG. 10, the lower end of the tube 33 (the end connected to the rubber plug 221) is disposed at a position corresponding to the opening 224 of the bottom plate 223. Thus, the tube 33 communicates with the inside of the container body 21.

[S3] Step of Sealing the Manufacturing Container After the void portion 11 of the container body 21 into which the mixed solution 10 has been injected is purged with the gas 3, the lid 22 is inserted into the opening (bottle neck) of the container body 21. Thereby, the mixture 10 and the gas 3 are sealed in the production container 20.

  Next, a syringe 40 filled with 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 conduit 321, and the conduit 321 and the tube 33 are communicated. Then, the injection needle 41 of the syringe 40 is inserted into the conduit 321, and then the gas 3 is further added from the syringe 40 into the manufacturing container 20 via the mininate valve 30 and the tube 33. Thereby, the inside of the manufacturing container 20 is pressurized.

  After that, simultaneously withdrawing the injection needle 41 from the conduit 321, the knob 341 on the right side of the handle 34 is pressed to the left to close the conduit 321, and the conduit 321 and the tube 33 are disconnected.

  Next, a 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 mininate valve 30 (upper side in the drawing) of the sealing portion 331, 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)).

  After that, 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 uniformly sized bubbles 1 can be stably manufactured in the manufacturing container 20. At the same time, a production container (closed container) 20 (bubble-containing container) containing a large amount of bubbles 1 of uniform size is obtained.

  In the present embodiment, in the step (S3), the inside of the manufacturing container 20 can be pressurized without directly puncturing the injection needle 41 into the rubber plug 221 of the lid 22. That is, in the present 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 sealing performance in the manufacturing container 20 can be improved. By improving the sealability in the manufacturing container 20, the bubble 1 can be more stably present in the mixture 10 in the finally obtained bubble-containing container. That is, the long-term storability of the bubble-containing container is further improved.

  The bubble producing method of the sixth embodiment produces the same operation and effect as the bubble producing methods of the first to fifth embodiments.

Seventh Embodiment
Next, a seventh embodiment of the method for producing a bubble of the present invention will be described.

  FIG. 11 is a perspective view for explaining the manufacturing container used in the seventh embodiment of the method for manufacturing a bubble of the present 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 manufacturing method of the bubble of the seventh embodiment will be described focusing on the difference from the manufacturing method of the bubble of the first to sixth embodiments, and the description of the same matters will be omitted.

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

  The production container 20 used in the present embodiment is, as shown in FIG. 11, the container body 21 used in the bubble manufacturing method of the sixth embodiment described above, the lid 22, the mininate valve 30, and the container body 21. And the tube 33 connecting the mininat valve 30. Furthermore, in the production container 20 of the present embodiment, the mininate valve 30 is provided on the lid 22 side (upper side in the drawing) than the liquid surface of the mixed solution 10.

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

  As shown in FIG. 11, the left end of the tube 33 is in communication with the internal space (void 11) of the container body 21, and the right end of the tube 33 is connected to the conduit 321 of the rubber plug 32. It is in communication. Therefore, also in the present embodiment, the conduit 321 and the internal space of the container body 21 are in communication via the tube 33.

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

  The bubble producing method of the seventh embodiment produces the same operation and effect as the bubble producing method of the first to sixth embodiments.

Eighth Embodiment
Next, an eighth embodiment of the method for producing a bubble of the present invention will be described.

  FIG. 12 is a cross-sectional view for explaining the manufacturing container used in the eighth embodiment of the method for manufacturing a bubble of the present 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”. Further, 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 manufacturing method of the bubble of the eighth embodiment will be described focusing on the difference from the manufacturing method of the bubble of the first to seventh embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of this embodiment is different from the manufacturing method of the bubbles 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). It is different. Specifically, in step (S4), the manufacturing container 20 is vibrated so as to reciprocate in substantially the horizontal direction of the manufacturing container 20 shown in FIG.

[S1] Preparation Step A production container 20 (a container for bubble production of the present embodiment) as shown in FIG. 12 is prepared.

  The manufacturing container 20 of the present embodiment includes a container main body 21 having a cylindrical portion 211 at the top, a rubber plug 23 for sealing the opening of the cylindrical portion 211, and weight portions 5 fixed to both ends of the container main body 21. Have.

  The container body 21 has a substantially cylindrical shape elongated in the horizontal direction. A cylindrical portion 211 protruding vertically upward from the upper surface of the container main body 21 is formed substantially at the center of the container main body 21. In the container body 21 of the present embodiment, only the opening of the cylindrical portion 211 is open to the outside. Further, screw grooves 212 are formed on both end sides of the container body 21.

  Although the rubber plug 23 is not particularly limited, for example, a rubber plug made of silicon can be used.

  The weight portion 5 has a disk-shaped flat plate portion 51 and a cylindrical portion 52 erected from the edge portion of the flat plate portion 51, and when the outer shape is a cross-sectional view, a member having a substantially C shape. It is. On the inner circumferential side of the cylindrical portion 52, a screw groove 521 that can be screwed into the screw groove 212 of the container main body 21 is formed. By screwing the screw groove 521 of the weight portion 5 into the screw groove 212 of the container main body 21, the weight portion 5 is attached to the container main body 21 with the flat plate portion 51 in close contact with each end of the container main body 21 Will be

  Similar to the bottom plate 223 of the lid 22 shown in FIG. 6 described above, such a weight 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 5 to the container body 21, the mass of both ends of the container body 21 can be increased. Thereby, in the step (S4), it is possible to further increase the size of the shock wave generated when the liquid mixture 10 collides with the portion (particularly, both end portions) fixed to the weight portion 5 of the container main body 21. As a result, fine bubbles 1 can be more easily and stably generated in the liquid mixture 10.

  From the viewpoint of high specific gravity and high corrosion resistance to the components of the mixed solution 10, it is preferable to use iron or an iron alloy such as stainless steel as the constituent material of the weight 5 among metal materials. Particularly preferred.

  Moreover, such a weight 5 is easily removable to the container body 21. The mass of the weight portion 5 can be appropriately adjusted by changing the constituent material and / or the size of the weight portion 5. By adjusting the mass of the weight portion 5, the size and the amount of generation of the bubble 1 generated in the liquid mixture 10 can be adjusted in the step (S4). That is, in the bubble manufacturing method of the present embodiment, bubbles 1 of various sizes and contents can be manufactured using the same manufacturing container 20. Therefore, since it is not necessary to prepare multiple types of manufacturing containers 20 with different sizes according to the bubble 1 of the target size and content, the productivity of the bubble-containing container is improved.

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

  Next, a syringe 40 filled with gas 3 is prepared. Then, the injection needle 41 of the syringe 40 is punctured into the rubber plug 23. Thereafter, additional gas 3 is added from the syringe 40 into the manufacturing container 20. Thereby, the inside of the manufacturing container 20 is pressurized. Thereafter, by removing the injection needle from the rubber plug 23, the production container 20 in which the inside of the production container 20 is pressurized by the gas 3 can be obtained.

[S4] Step of Vibrating the Manufacturing Container Next, the manufacturing container 20 is vibrated so that the mixed solution 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 such that the manufacturing container 20 substantially reciprocates 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 conditions similar to 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 performing the steps (S5) to (S10) in the same manner as the third embodiment. A large amount of bubbles 1 of uniform size can be stably produced in the inside. At the same time, a production container 20 containing a large amount of uniformly sized bubbles 1 is obtained.

  The bubble producing method of the eighth embodiment produces the same action and effect as the bubble producing methods of the first to seventh embodiments.

The Ninth Embodiment
Next, a ninth embodiment of the method for producing a bubble of the present invention will be described.

  FIG. 13 is a cross-sectional view for explaining the manufacturing container used in the ninth embodiment of the method for manufacturing a bubble of the present invention. FIG. 13 (a) shows the manufacturing container in the disassembled state, and FIG. 13 (b) shows the manufacturing container in the assembled state.

  In the following description, the upper side in FIGS. 13 (a) and 13 (b) is referred to as “upper”, and the lower side in FIGS. 13 (a) and 13 (b) is referred to as “lower”. Also, 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 manufacturing method of the bubble of the ninth embodiment will be described focusing on differences from the manufacturing method of the bubble of the first to eighth embodiments, and the description of the same matters will be omitted.

  The bubble manufacturing method of this 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 as shown in FIGS. 13 (a) and 13 (b). It is the same as the manufacturing method of the bubble using the manufacturing container 20 shown to 12. That is, the container main body 21 is comprised by the member which makes the cylindrical shape which both ends opened outside.

  When using the manufacturing container 20 of such a configuration, the weight portion 5 is attached to the container main body 21 by screwing the screw groove 521 of the weight portion 5 with the screw groove 212 of the container main body 21 first. Note that, as shown in FIG. 13B, in the 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 may be disposed between the flat plate portion 51 and each end of the container body 21 to enhance the adhesion between the weight portion 5 and the container body 21. Thereby, the sealing performance of the manufacturing container 20 can be improved.

  Then, the 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 by sucking the bubble-containing mixture after the rubber plug 23 is punctured with the injection needle of the syringe. Moreover, in such a configuration, it is possible to remove the weight portion 5 from the container main body 21 and directly take out the bubble-containing mixed solution from the bubble-containing container without using a syringe.

  The bubble producing method of the ninth embodiment produces the same action and effect as the bubble producing methods 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 a cross-sectional view for explaining the manufacturing container used in the tenth embodiment of the method for manufacturing a bubble of the present invention. FIG. 14 (a) shows the manufacturing container in the disassembled state, and FIG. 14 (b) shows the manufacturing container in the assembled state. FIG. 15 is a view for explaining the position of the opening formed in the lid of the production container shown in FIG. 14 (b). Fig.15 (a) is a figure for demonstrating the state before puncturing the injection needle of a syringe in a rubber plug, and FIG.15 (b) is a bottom plate after clamping a needle from the rubber plug. It is a figure for demonstrating the state clamped to the part.

  In the following description, the upper side in FIGS. 14 (a) and 14 (b) is referred to as “upper”, and the lower side in FIGS. 14 (a) and 14 (b) is referred to as “lower”. 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 manufacturing method of the bubble of the tenth embodiment will be described focusing on the difference from the manufacturing method of the bubble of the first to ninth embodiments, and the description of the same matters will be omitted.

  The manufacturing method of the bubble of this embodiment is the same as the manufacturing method of the bubble 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 production container 20 as shown in FIG. 14 (b) is prepared.

  The manufacturing container 20 of the present embodiment has a container main body 21 similar to the manufacturing container 20 of the first embodiment described above, and a lid 22.

  In the present embodiment, the lid 22 includes a bottom plate portion 223 fixed to the bottle mouth of the container main body 21, a rubber plug 221 disposed on the opposite side of the bottom plate portion 223 to the container main body 21, and a rubber plug 221 And a fastening portion 222 for fixing to 223.

  The bottom plate portion 223 has a disk-shaped flat plate portion 225 and a cylindrical portion 226 erected from the edge portion of the flat plate portion 225, and when the outer shape is a cross-sectional view, a member having a substantially C shape. It is. In the present embodiment, the shape of the bottom plate portion 223 (flat plate portion 225) in plan view (upper surface view) and the shape of the rubber plug 221 are substantially equal, and have substantially the same diameter. Moreover, the screw groove mutually formed screwably is formed in the inner peripheral surface of the cylindrical part 226, and the outer peripheral surface by the side of the bottle opening of the container main body 21, By screwing these, The bottom plate portion 223 (flat plate portion 225) is fixed in a state in which the bottom plate portion 223 (flat plate portion 225) is in close contact with the bottle opening of the container body 21.

  Further, in the bottom plate portion 223, an opening 224 having a size allowing insertion of the injection needle of the syringe is formed at a position separated by a predetermined distance from the center in plan view (top view). That is, as shown in FIG. 15A, in a plan view of the lid 22, the center C of the rubber plug 221 and the opening 224 of the bottom plate 223 are misaligned.

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

  As the rubber plug 221, a rubber plug similar to the rubber plug 221 used in the method of manufacturing the bubble 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 223 in a state where 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 to be mutually screwable are respectively formed on the outer peripheral surfaces of the tightening portion 222 and the bottom plate portion 223 (flat plate portion 225), and the rubber plug 221 is formed by screwing these together. Is fixed in close contact with the bottom plate 223 (flat plate 225).

[S3] Step of Sealing the Manufacturing Container First, after the void portion 11 of the container body 21 into which the mixed solution 10 is injected is purged with the gas 3, the lid 22 is attached to the opening (bottle neck) of the container body 21. (State shown in FIG. 15 (a)). Thereby, the mixture 10 and the gas 3 are sealed in the production container 20.

  In the state shown in FIG. 15A, the injection needle of the syringe is inserted into the mark X of the rubber plug 221, and the injection needle is inserted through the opening 224 of the bottom plate 223. Then, after gas 3 is further added from the syringe into the manufacturing container 20 and the inside of the manufacturing container 20 is pressurized, the injection needle is removed from the rubber plug 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 fastening the fastening 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, the position of the through hole 227 formed in the rubber plug 221 and the position of the opening 224 of the bottom plate 223 are deviated by the puncturing of the injection needle in plan view (see FIG. 15B). As a result, the opening 224 of the bottom plate 223 is closed by the rubber plug 221, and the manufacturing container 20 in which the inside of the manufacturing container 20 is pressurized by the gas 3 can be obtained.

  In the present embodiment, since there is no portion that communicates the inside and the outside of the manufacturing container 20, the sealing performance in the manufacturing container 20 can be improved. By improving the sealability in the manufacturing container 20, the bubble 1 can be more stably present in the mixture 10 in the finally obtained bubble-containing container. That is, the long-term storability of the bubble-containing container is further improved.

As described above, the rubber plug 221 is compressed to the bottom plate portion 223 side by tightening the tightening portion 222 to the bottom plate portion 223. The thickness of the rubber plug 221 in a state before tightening the tightening portion 222 to the bottom plate portion 223 is t ₁ (mm), and the thickness of the rubber plug 221 in a state where the tightening portion 222 is tightened to the bottom plate portion 223 is t ₂ When (mm), the compression ratio ((t ₁ -t ₂ ) / t ₁ × 100) of the rubber plug 221 is preferably 5 to 60%, and more preferably 10 to 30%. Thus, the adhesion between the rubber plug 221 and the bottom plate 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 sealability in the manufacturing container 20 can be further improved.

  Also in the present embodiment, a large amount of bubbles 1 of uniform 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 uniformly sized bubbles 1 is obtained.

  The bubble producing method of the tenth embodiment produces the same operation and effect as the bubble producing methods of the first to ninth embodiments.

As mentioned above, although the manufacturing method of the bubble of the present invention and the container for bubble production were explained based on the embodiment of illustration, the present invention is not limited to this, and each process can exhibit the same function. It can replace with an optional step.
For example, any configuration of the first to tenth embodiments can be combined.

  Here, in order to clarify the influence of the volume of the gas 3 sealed in the production container 20 and the rotation speed of the production container 20 on the particle size and content of the bubble 1 generated in the mixed liquid 10, the following I did an experiment.

  First, the relationship between the rotation speed of the production container 20 and the particle size and content of the bubble 1 generated in the liquid mixture 10 was examined.

(Method of manufacturing bubble)
[Preparation process]
First, 120 μl of an albumin solution containing 25 mg / ml of albumin (CSL Behring, Albuminer 25%) 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 that of the manufacturing container 20 shown in FIG.

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

[Step of sealing the container]
Next, after the space in the vial into which the mixed solution was injected was purged with perfluorobutane, a lid having the same shape as the lid 22 shown in FIG. 3 was inserted into the mouth of the vial. Next, a syringe filled with perfluorobutane was prepared. The rubber stopper of the lid was punctured with a syringe needle of a syringe, and an additional 2 ml of perfluorobutane was added from the syringe into the vial. Thus, the pressure in the vial was 2 atm, and the vial was sealed to obtain a sealed vial.

[Step of vibrating the container]
Next, two sealed vials as described above were prepared. One closed vial was shaken at a rotation speed of 5000 rpm for 30 seconds, and the other closed vial was shaken at a rotation speed of 6500 rpm for 30 seconds using Precellys (a high-speed cell disruption system) manufactured by Bertin Technologies. At that time, the closed vial was 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 vibration width in the vertical direction of the closed vial was 40 mm, and the horizontal vibration width of the closed vial was 20 mm.

[Step of allowing the container to settle]
After shaking, the sealed vial was allowed to stand to obtain a bubble-containing container.

(Particle size distribution measurement)
The mixed solution containing bubbles (bubble-containing mixed solution) was taken out by a syringe from the bubble-containing container obtained as described above, and then, using the bubble measuring apparatus (nanoparticle analysis system nanosight), the mixed solution The particle size distribution of the bubbles contained in was measured. The results are shown in FIG.

  FIG. 16 (a) is a graph showing the particle size distribution of the bubbles when the bubbles are produced at rotation speeds of 5000 rpm and 6500 rpm. FIG. 16B is a partially enlarged view of the graph shown in FIG. 16A in which the horizontal axis is in the range of 0 to 700 nm.

  As shown in FIG. 16 (a), by vibrating the closed vial at 6500 rpm, the content of bubbles in the mixture can be significantly increased as compared to the case where the closed vial is vibrated at 5000 rpm. did it. In particular, the content of bubbles smaller than about 600 nm in particle size is three to five times more in the case of vibrating the closed vial at 6500 rpm, as compared with the case of vibrating the closed vial at 5000 rpm became.

  Even when the rotation speed of the closed vial is 5000 rpm, the content of bubbles in the mixture can be increased to a certain extent by prolonging the vibration time. However, the content of the bubbles is less than that at a rotational speed of 6500 rpm.

  Further, as shown in FIG. 16B, when the closed vial was vibrated at 6500 rpm, a large number of minimal bubbles with a particle diameter 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, depending on the rotation speed of the closed vial, the magnitude of the pressure of the shock wave generated when the mixture collides with the vial changes. The magnitude of this shock wave pressure is a major factor in determining the particle size and content of bubbles generated in the mixture. In the case of stirring using a general stirrer or vibration at a rotational speed lower than 5000 rpm, such a shock wave is not generated or, if generated, the amount generated is small. Therefore, bubbles having a sufficiently small particle size as in the present invention can not be generated in the mixture at a sufficient content.

FIG. 17 shows the result of analysis of the particle size distribution graph shown in FIG.
FIG. 17 (a) is a graph showing the relationship between the number of revolutions of a closed vial and the average particle size of bubbles. FIG. 17 (b) is a graph showing the relationship between the number of revolutions of the closed vial and the content of bubbles.

As shown in FIG. 17 (a), when the closed vial is vibrated at 6500 rpm, the average particle size of the generated bubbles is about 80 nm smaller than when the closed vial is vibrated at 5000 rpm. Become. Further, as shown in FIG. 17 (b), when the closed vial is vibrated at 6500 rpm, the content of the generated bubbles is 9 ×, as compared to the case where the closed vial is vibrated at 5000 rpm. 10 ⁷ particles / ml or less. This result also shows that when the closed vial is vibrated at 6500 rpm, it is possible to generate a large amount of bubbles having a smaller particle diameter 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 bubble 1 generated in the mixture 10 was examined.

(Method of manufacturing bubble)
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, In the same manner as in Example 1, a bubble-containing container was obtained.

  For each sealed vial, the volume (ml) of the gas (perfluorobutane) to be sealed, the pressure (atm) in the sealed vial and the number of revolutions (rpm) of the sealed vial in the step of vibrating the container It is shown in Table 1.

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

  FIG. 18 (a) is a graph showing the relationship between the volume of gas sealed in a sealed vial and the average particle size of bubbles. FIG. 18 (b) is a graph showing the relationship between the volume of gas sealed in a sealed vial and the content of bubbles.

  As shown in FIG. 18 (a), even if the number of revolutions at which the sealed vial is vibrated is the same, increasing the pressure in the sealed vial reduces the average particle size of the generated bubbles. Specifically, when the pressure in the closed vial is 2 atm, the average particle size of the generated bubbles is about 100 nm smaller than when the pressure in the closed vial is 1.2 atm. The

  In addition, as shown in FIG. 18 (b), the bubble content was increased by increasing the pressure in the closed vial. In particular, when the pressure in the closed vial was 2 atm, the content of the bubble was more than twice as high as in the case where the pressure in the closed vial was 1.2 atm.

  By increasing the pressure in the closed vial, it is believed that the pressure of the shock wave generated when the mixture and the vial collide collide with each other to greatly affect the particle size and content of the bubbles generated. Therefore, even if the number of revolutions at which the closed vial is vibrated is the same, the average particle size of the generated bubbles changes according to the pressure in the closed vial. Also, by increasing the pressure in the closed vial, a large amount of gas is taken into the mixture. Therefore, the content of bubbles generated in the mixture 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 an albumin solution (CSL Behring 25% Albuminer) 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 that of the manufacturing container 20 shown in FIG.

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

[Step of sealing the container]
Next, after the space in the vial into which the mixed solution was injected was purged with perfluorobutane, a lid having the same shape as the lid 22 shown in FIG. 3 was inserted into the mouth of the vial. Next, a syringe filled with perfluorobutane was prepared. The rubber stopper of the lid was punctured with a syringe needle of a syringe, and an additional 2 ml of perfluorobutane was added from the syringe into the vial. Thus, the pressure in the vial was 2 atm, and the vial was sealed to obtain a sealed vial.

[Step of vibrating the container]
Next, the sealed vial was shaken at 7,000 rpm for 30 seconds using Precellys manufactured by bertin Technologies. At that time, the closed vial was 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 vibration width in the vertical direction of the closed vial was 40 mm, and the horizontal vibration width of the closed vial was 20 mm.

[Step of allowing the container to settle]
After shaking, the sealed vial was allowed to stand to obtain a bubble-containing container. In addition, the mixed solution containing bubbles (bubble-containing mixed solution) was taken out with a syringe, and the bubble size was confirmed using a 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>
0.2 μg of the mixed solution obtained in Example 3 is added to a Petri dish cultured with cerebrovascular pericytes (pericyte) (product code: C-12980, manufactured by Takara Bio Inc.) to obtain cerebrovascular pericytes. The culture medium was obtained. Pericyte is known as a very difficult cell for gene transfer.

  Four samples of such cerebrovascular pericyte cultures were prepared. These samples were irradiated with ultrasonic waves of frequency: 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 cerebrovascular pericyte culture medium at 37 ° C. for 48 hours was observed with a fluorescence microscope.

FIG. 19 is a fluorescence microscope image of a cerebrovascular pericyte culture medium cultured at 37 ° C. for 48 hours, and FIG. 19 (a) is an image of a sample irradiated with ultrasonic waves at an irradiation intensity of 0.6 W / cm ² FIG. 19 (b) 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 microscope image of a cerebrovascular pericyte culture medium cultured at 37 ° C. for 48 hours, and FIG. 20 (a) shows an ultrasonic intensity of a sample irradiated at an irradiation intensity of 0.9 W / cm ² . It is an image and FIG. 20 (b) 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) and (b) and FIGS. 20 (a) and 20 (b), in each of the samples irradiated with ultrasonic waves at any irradiation output, a region colored in green was confirmed. This indicates that in each sample, green fluorescent protein (GFP) is expressed in cerebrovascular pericytes. Therefore, it was shown that in any of the samples, the bubbles were ruptured by the ultrasonic irradiation, and the GFP gene contained in the bubbles was introduced into the cerebrovascular pericytes.

  DESCRIPTION OF SYMBOLS 1 ... Bubble 2 ... Outer shell 3 ... Gas 4 ... Drug 10 ... Mixed liquid 11 ... Void part 20 ... Manufacturing container 21 ... Container main body 211 ... Cylindrical part 212 ... Thread groove 22 ... Lid 221 ... Rubber stopper 222 ... Tightening part 223 ... Bottom plate portion 224: Opening portion 225: Flat portion 226: Cylindrical portion 227: Through hole 23: Rubber plug 30: Mininate valve 31: Bubble body 311, 312: Through hole 32: Rubber plug 321: Pipe path 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 part 52: Tubular part 521: Threaded groove X: Mark C: Center

Claims (16)

Injecting a solution containing the target substance to a predetermined height of the container;
Vibrating the container at a number of revolutions of 5000 rpm or more so that the solution repeatedly collides with the inner surface of the container with the pressure in the container being greater than 1.0 atm.
Vibrating the container includes vibrating the container by rotational movement or rotational movement and reciprocating movement of the container in the vertical direction,
When the height of the container is X (mm), the vibration width in the vertical direction of the container is 0.7 X to 1.5 X (mm).   The method for producing a bubble according to claim 1, wherein the step of vibrating the container includes the step of generating a shock wave by causing the solution to collide with the inner surface of the container.   The 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 reciprocating and / or rotational movement in the horizontal direction of the container,
The method for manufacturing a bubble 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 velocity between the container and the solution at the time of colliding the solution with the inner surface of the container is 40 km / h or more. The manufacturing method of the bubble as described in.   The method for producing a bubble according to any one of claims 1 to 5, wherein the step of vibrating the container is performed in a state where the pressure is 1.5 to 10 atm.   The predetermined height is 0.2 ≦ Y /, where Y (mm) is the height of the liquid surface of the solution in the container in a state where the container into which the solution is injected is allowed to stand horizontally. The method for producing a bubble according to any one of claims 1 to 6, wherein the relationship of X? 0.7 is satisfied.   The process according to any one of claims 1 to 7, further comprising the step of revibrating the container at a rotational speed of 5,000 rpm or more after changing the pressure in the container after the step of vibrating the container. Method.   The method for producing a bubble according to claim 8, wherein the step of revibrating the container is performed such that the pressure is 1 to 10 atm higher than the pressure in the step of vibrating the container.   The method for producing a bubble according to claim 8 or 9, wherein the step of revibrating the container is performed at a rotation number different from the rotation number in the step of vibrating the container.   The method for producing a bubble according to any one of claims 1 to 10, wherein the step of injecting the solution into the container and the step of vibrating the container are performed so as to keep the temperature of the solution constant.   The method for producing a bubble according to any one of claims 1 to 11, wherein the target substance comprises an outer shell material constituting an outer shell of the bubble.   The outer shell material is an amphiphilic material containing at least one of a protein, a polycationic lipid, a phospholipid, a higher fatty acid, a saccharide, a sterol, a surfactant, and a natural and synthetic polymer. Method for producing the described bubble.   The production of the bubble 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 bilayer of molecules of the amphiphilic material. Method.   The method for producing a bubble according to any one of claims 1 to 14, wherein the target substance comprises a drug.   The method for producing a bubble according to any one of claims 1 to 15, wherein an average particle size of the bubble is 10 nm to 1000 μm.

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