Method for producing nanobubble

Abstract

To construct a method for readily preparing a highly safe bubble formulation.SOLUTION: The present invention provides a method for producing nanobubbles with shells composed of peptides or proteins, including the step of lyophilizing a mixture of microbubbles and nanobubbles with shells composed of peptides or proteins.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing nanobubbles, and more particularly to a method for producing nanobubbles having a shell made of a peptide or protein. [Background technology]

[0002] According to the ISO definition, bubbles with a diameter of less than 100 μm are called fine bubbles (FB). Fine bubbles with a diameter of 1 μm to 100 μm are called "microbubbles," and bubbles with a diameter of less than 1 μm are called ultra-fine bubbles (UFB, also called "nanobubbles"). Applications of microbubbles and nanobubbles are being considered in various fields, one of the main fields being medical research and healthcare. In the medical research and healthcare fields, microbubbles and nanobubbles are used as tools for testing, diagnosis, and treatment.

[0003] One of the medical applications of microbubbles is as an ultrasound contrast agent in ultrasound diagnostic imaging. Microbubbles have a soft surface and resonate with ultrasound, so they have strong echogenicity. Taking advantage of this property, microbubbles are applied to ultrasound diagnostic imaging.

[0004] Another application of microbubbles in the medical field is a drug delivery system (DDS). When ultrasonic waves are applied to microbubbles present in liquid, the microbubbles expand and contract repeatedly in synchronization with the frequency, absorbing surrounding gas molecules, growing, and finally bursting. This series of events is called "acoustic cavitation." Microbubbles that burst as a result of acoustic cavitation generate micro liquid jets, which can be used for a variety of purposes. For example, the present inventors have reported that the efficacy of drugs can be enhanced by using micro liquid jets (Non-Patent Document 1).

[0005] In addition, by generating a micro liquid jet caused by the bursting of microbubbles near a cell, the micro liquid jet can temporarily create holes in the cell membrane, and a technology (called "sonoporation") has been developed that uses such holes to deliver drugs into the cell.

[0006] The strength of sonoporation is that it can deliver various therapeutic modalities, such as small molecular compounds, genes (e.g., DNA, mRNA, siRNA, etc.), antibodies, and peptides, into cells. A particular advantage of sonoporation-based DDS is that it can easily deliver macromolecules into cells. For example, other than sonoporation, the common means of delivering macromolecules such as plasmid DNA into cells are chemical carriers such as viral vectors, liposomes, and polymeric micelles. However, these methods require the preparation of chemical carriers to carry the macromolecules in addition to the macromolecules to be delivered, which makes the process complicated and increases costs. On the other hand, sonoporation can deliver macromolecules into cells in a carrier-free manner, making it possible to create a simple and low-cost DDS.

[0007] Another advantage of drug delivery using microbubbles and ultrasound is that the delivery site can be precisely controlled. By using MRI-guided focused ultrasound (magnetic resonance-guided FUS: MRgFUS), the ultrasound irradiation site can be controlled in millimeters. One application example is BBB opening (Blood Brain Barrier Opening: BBBO). Cerebral arteries have a structure called the blood brain barrier (BBB), in which vascular endothelial cells are tightly bound to each other, which restricts the delivery of substances into the brain. In BBBO, microbubbles are administered into blood vessels along with drugs, and ultrasound is irradiated deep into the brain using MRgFUS. By contracting, expanding, and bursting the microbubbles at the target site, and temporarily opening the BBB, drugs can be delivered into the brain.

[0008] In recent years, applications of nanobubbles, which are even finer than microbubbles, have been actively studied. Although the physical properties of nanobubbles have not yet been fully elucidated due to the fact that they are very small and difficult to observe, it has been reported that they can be applied to ultrasound contrast agents and DDS, including sonoporation, in the same way as microbubbles. The present inventors have reported that mRNA can be delivered into cells in a carrier-free manner by sonoporation using nanobubbles (Non-Patent Document 2).

[0009] Microbubbles and nanobubbles have been improved in various ways. For example, air was used as the gas contained in the bubbles in the early stages, but later, gases with high molecular weights and low solubility such as perfluoropropane (C3F8), perfluorobutane (C4F10), and sulfur hexafluoride (SF6) were used, which resulted in a significant improvement in the stability of the bubbles and contributed to the extension of the duration of the contrast effect. In addition, human serum albumin was used as the bubble shell in the early stages, but later, poly(lactic acid-glycolic acid) (PLGA), palmitic acid, and phospholipids were used as shell components, contributing to the diversification of the properties of microbubbles. Furthermore, it is now possible to improve the retention performance of microbubbles by applying chemical modifications such as PEG to the surface of the bubble shell, and to impart targeting to microbubbles by binding molecules with affinity for specific targets such as antibodies to the surface of the bubble shell.

[0010] As mentioned above, microbubbles and nanobubbles have been extensively studied from various perspectives, and a great deal of academic knowledge has been accumulated. However, due to issues such as safety and cost, there are currently only a few types of bubble preparations that can actually be used in clinical practice, and their applications are extremely limited.

[0011] One of the reasons why it is difficult to market bubble preparations is that it is difficult to make the bubble size uniform. For example, when preparing a nanobubble solution, even a small number of microbubbles may be mixed in, which may have a significant effect on the physical properties of the entire bubble solution. Theoretically, the gas volume contained in a bubble is proportional to the cube of its hollow radius. If the volume of a nanobubble with a hollow radius of 100 nm is compared with that of a microbubble with a hollow radius of 1 μm, which is 10 times larger, the gas volume contained in the latter is equivalent to 1000 times that of the former. Therefore, even if a small number of microbubbles are mixed in a nanobubble solution, the impact on its physical and physiological actions is extremely large. In order to make the action of the nanobubble solution uniform, it is important to remove as many microbubbles as possible that are mixed in the solution. Therefore, there is a strong need to establish a new bubble manufacturing method that can prepare a highly safe bubble preparation at low cost. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] K. Tachibana et al., Circulation. 1995 Sep 1;92(5):1148-50. [Non-Patent Document 2] H. Kida et al., Front Pharmacol. 2022 Jun 1;13:855495. Summary of the Invention [Problem to be solved by the invention]

[0013] In light of this background, an object of the present invention is to develop a method for easily preparing a highly safe bubble preparation (particularly a nanobubble preparation). [Means for solving the problem]

[0014] As a result of intensive research into the above-mentioned problems, the present inventors have discovered that by freeze-drying an aqueous solution containing microbubbles and nanobubbles having a shell made of highly safe albumin, (1) It is possible to selectively destroy microbubbles and maintain only nanobubbles. (2) Freeze-dried nanobubbles can be stored for long periods at temperatures between 4 and 25°C. (3) We found that nanobubbles after long-term storage can be regenerated by redissolving them in distilled water or the like, and can be used as ultrasound contrast agents or for sonoporation, just like the nanobubbles before freeze-drying.

[0015] In the production of bubble preparations, freeze-drying is a method generally used for bubbles having a bubble shell made of highly flexible phospholipids, and it was thought that freeze-drying would collapse bubbles having a relatively low flexibility bubble shell such as an albumin shell. Due to the existence of such technical common knowledge, freeze-drying has hardly been applied to bubbles having an albumin shell. Therefore, the inventors' discovery that freeze-drying treatment can selectively collapse albumin shell microbubbles while maintaining albumin shell nanobubbles, and that freeze-dried albumin shell nanobubbles can be stored for a long period of time in a temperature range from refrigeration to room temperature, was extremely surprising. The inventors further conducted research based on such findings and have completed the present invention. That is, the present invention is as follows.

[0016] [1] A method for producing dry nanobubbles having a peptide or protein shell, comprising the step of freeze-drying a mixture of microbubbles and nanobubbles having a peptide or protein shell. [2] The method for producing a mixture of microbubbles and nanobubbles having a shell made of a peptide or protein is not subjected to a separation step utilizing buoyancy. [3] The manufacturing method according to [1] or [2], wherein the freeze-drying is vacuum freeze-drying. [4] The method according to any one of [1] to [3], wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme. [5] A method for removing microbubbles from a mixture of peptide or protein shelled microbubbles and nanobubbles, comprising the step of freeze-drying the mixture of peptide or protein shelled microbubbles and nanobubbles. [6] The method according to [5], characterized in that the mixture of microbubbles and nanobubbles having a shell made of a peptide or protein is not subjected to a separation step utilizing buoyancy. [7] The method according to [5] or [6], wherein the freeze-drying is vacuum freeze-drying. [8] The method according to any one of [5] to [7], wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme. [9] A method for reducing the size of nanobubbles having a shell made of a peptide or protein, the method comprising the step of freeze-drying nanobubbles having a shell made of a peptide or protein.

[10] The method for micronization described in [9], wherein the freeze-drying is vacuum freeze-drying.

[11] The method for micro-sizing according to [9] or

[10] , wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme.

[12] A dry nanobubble having a shell made of a peptide or protein, or a composition containing the same.

[13]

[12] The composition according to claim 1, wherein the residual rate of microbubbles contained in the composition is 1% or less, and the average particle size of the nanobubbles is within the range of 1 to less than 1,000 nm.

[14] The nanobubbles or composition according to

[12] or

[13] , wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme. Effect of the Invention

[0017] Although no freeze-dried preparation of nanobubbles having an albumin shell has existed so far, it can be prepared by the present invention. In addition, in order to separate a mixture of nanobubbles from a mixture of microbubbles and nanobubbles having an albumin shell, the difference in buoyancy between the two has been generally used. However, it is very difficult to separate only nanobubbles with high purity from a mixture of microbubbles and nanobubbles by separation based on the difference in buoyancy, and as a result, it is not easy to prepare a high-purity albumin-shelled nanobubble composition that is substantially free of microbubbles as impurities. However, according to the present invention, high-purity albumin-shelled nanobubbles can be extremely easily prepared by freeze-drying alone, without performing separation using the buoyancy of the microbubbles, such as standing or centrifugation. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of the sonication or sonoporation method for nanobubbles in a 96-well plate. (A) Sonication method for nanobubbles. Ultrasonic irradiation for nanobubbles. (BF) Sonoporation method. (B) Removal of incubation medium from wells of a 96 multi-well plate seeded with HSC-2 cells. (C) Filling of wells with a solution containing nanobubbles. (D) Gene transfection by sonication. (E-1) Aspirating sonicated medium. (E-2) Adding fresh incubation medium. (F) After 24 hours of incubation, harvesting the supernatant for reporter assay. (G) Arrangement of wells seeded with cells (indicated in color) and sonication area (inside dashed circle) on a 96-well plate. [Diagram 2]Figure 2 shows the flow phantom and the measurement setup: NBs(+): solution with nanobubbles, NBs(-): solution without nanobubbles. [Diagram 3] FIG. 3 shows the appearance of the lyophilized albumin solution (A) with and (B) without bubbling. [Figure 4] Figure 4 shows SEM images of the lyophilized albumin solution with and without bubbling. The lyophilized albumin solution with bubbling at low magnification (A: ×1,000), high magnification (B: ×10,000), and ultra-high magnification (C: ×30,000). The lyophilized albumin solution without bubbling at low magnification (D: ×1,000), high magnification (E: ×10,000), and ultra-high magnification (F: ×30,000). [Diagram 5] Figure 5 shows the change in bubble properties in solution before and after freeze-drying. (A) Comparison of bubble concentration and bubble distribution in FCM measurement. (B) Comparison of bubble concentration and particle size in NTA measurement. (C) Comparison of bubble concentration and bubble suspended mass in RMM measurement. LPh(-): solution before freeze-drying, LPh(+): solution after freeze-drying. [Figure 6] Figure 6 shows the change in bubble properties in a solution after freeze-drying by sonication. (A) Comparison of bubble concentration and size distribution in FCM measurements. (B) Comparison of bubble concentration and particle size in NTA measurements. (C) Comparison of bubble concentration and bubble suspended mass in RMM measurements. [Figure 7] Figure 7 shows the detection of echogenicity changes in a flow phantom tube with solutions with or without nanobubbles regenerated from a lyophilized material. (A) B-mode only. (B) B-mode with Coded Harmonic Angio. (C) B-mode with Amplitude Modulation. NBs(-): solution without nanobubbles, NBs(+) solution with nanobubbles. [Figure 8]Figure 8 shows the mRNA transfection efficiency (A) and cell viability (B) by sonoporation using nanobubbles regenerated from lyophilized material. NBs(+): solution with nanobubbles, NBs(-): solution without nanobubbles. (***p<0.001, ns: no significant difference) (N=3). [Figure 9] 9 is a diagram showing the temperature change of a 0.06% human serum albumin aqueous solution during the bubbling process. The room temperature at the time of measurement was 18.9° C. The area indicated in gray: vibration process. [Figure 10] FIG. 10 is a diagram showing a comparison of the remaining concentration of microbubbles in a solution before and after vacuum freeze-drying. [Figure 11] FIG. 11 is a graph showing the change in bubble concentration when freeze-dried nanobubble aggregates were redissolved in 1.2 mL and 3.6 mL of pure water. [Figure 12] FIG. 12 is a graph showing the biofilm formation inhibitory effect of nanobubbles regenerated from a freeze-dried product. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail below. In this specification, "microbubbles" refers to bubbles having a diameter of 1 to less than 100 μm, and "nanobubbles" refers to bubbles having a diameter of less than 1 μm. In this specification, microbubbles may be referred to as "MB" or "MBs", and nanobubbles may be referred to as "NB" or "NBs".

[0020] 1. Nanobubble production method The present invention provides a method for producing nanobubbles having a shell made of a peptide or protein (hereinafter sometimes referred to as the "production method of the present invention"), which comprises a step of freeze-drying a mixture of microbubbles and nanobubbles having a shell made of a peptide or protein.

[0021] The microbubbles and nanobubbles used in the production method of the present invention are peptides or proteins whose shells are composed of amino acids (preferably natural amino acids).

[0022] The peptide constituting the shell of the microbubbles or nanobubbles used in the production method of the present invention may generally have a linear or branched amino acid sequence of 2 to 36 residues, preferably 4 to 24 residues.

[0023] In one embodiment, the peptide may be hydrophobic, and a consecutive sequence of isoleucine may be particularly preferable as the hydrophobic sequence. Furthermore, the peptide may contain a non-natural amino acid or may be modified in part.

[0024] In another embodiment, the peptide may have a hydrophilic amino acid sequence (2 to 8 residues) at either or both of the C-terminus and N-terminus. Alternatively, in another embodiment, the peptide may have a hydrophobic amino acid sequence (2 to 8 residues) at either or both of the C-terminus and N-terminus. In a preferred embodiment, the peptide may have a hydrophilic amino acid sequence (2 to 8 residues) at one end and a hydrophobic amino acid sequence (2 to 8 residues) at the other end. It is presumed that a peptide having this structure becomes amphiphilic, functions as a surfactant, and is located at the air-liquid interface.

[0025] Although not wishing to be bound by theory, it is known that highly hydrophobic amino acid sequences improve the efficiency of intracellular delivery of drugs or genes. Therefore, when nanobubbles prepared by the production method of the present invention are used for the purpose of DDS, it may be desirable to increase the ratio of hydrophobic amino acids in the peptide or protein that constitutes the shell of the nanobubbles. Nanobubbles consisting of such highly hydrophobic amino acid sequences can be prepared by a method known per se. One example is a method of modifying the amino acid sequence of a peptide or protein so that it has a consecutive sequence of phenylalanine or isoleucine, but is not limited to this.

[0026] The peptides or proteins constituting the outer shells of microbubbles or nanobubbles used in the production method of the present invention are not particularly limited as long as they are peptides or proteins made of amino acids (preferably natural amino acids), can constitute the outer shells of the bubbles, and can achieve the desired effects of the present invention. Examples include albumin, immunoglobulin G, and lysozyme, or fragments thereof that can constitute the outer shells of the bubbles.

[0027] The albumin may be albumin derived from any animal, and is preferably human serum albumin, bovine serum albumin, or ovalbumin. It may also be a fragment of albumin, so long as it can form the outer shell of the bubble.

[0028] Immunoglobulin G (hereinafter sometimes referred to as IgG) may be IgG from any animal, but is preferably IgG from a mammal, and more preferably IgG from a human. IgG may be humanized IgG. In addition, IgG may be of any subtype. Specifically, IgG may be any of IgG1, IgG2, IgG3, or IgG4.

[0029] The lysozyme may be of any animal origin, but is preferably of human origin.

[0030] In a preferred embodiment of the production method of the present invention, the microbubbles or nanobubbles may be microbubbles or nanobubbles having a shell made of albumin, more preferably, the microbubbles or nanobubbles may be microbubbles or nanobubbles having a shell made of human serum albumin.

[0031] As described above, the microbubbles and nanobubbles used in the production method of the present invention have a shell that is a peptide or protein composed of amino acids (preferably natural amino acids). In other words, the microbubbles and nanobubbles used in the production method of the present invention do not substantially contain components other than the peptide or protein composed of amino acids as their outer shell.

[0032] Examples of components other than peptides or proteins composed of such amino acids include lipids (particularly phospholipids), surfactants, and PLGA (polylactic acid-co-glycolic acid). The microbubbles and nanobubbles used in the production method of the present invention do not contain these components as outer shells.

[0033] Examples of lipids include the following lipids or derivatives thereof: (1) Phosphatidylethanolamine (e.g., dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), etc.); (2) phosphatidylcholine (e.g., distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dioleylphosphatidylcholine (DOPC), etc.); (3) phosphatidylserine (e.g., dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleyl phosphatidylserine (DOPS), etc.); (4) Phosphatidic acids (e.g., dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA), etc.); (5) phosphatidylglycerol (e.g., distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylglycerol (DOPG), etc.); (6) Phosphatidylinositol (e.g., dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleylphosphatidylinositol (DOPI), etc.).

[0034] Examples of the surfactant include the following surfactants or derivatives thereof: (1) Anionic surfactants (e.g., sodium lauryl sulfate, etc.); (2) nonionic surfactants (e.g., glycerin fatty acid esters (e.g., glycerin monostearate, etc.), sucrose fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan monostearate, sorbitan monolaurate, etc.), polyglycerin fatty acid esters, polyoxyethylene (hydrogenated) castor oil, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan laurate esters (e.g., polysorbate 20, etc.), polyoxyethylene sorbitan oleate esters (e.g., polysorbate 80, etc.), etc.), polyethylene glycol fatty acid esters, polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether, etc.), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene cetyl ether, etc.), polyoxyethylene alkyl phenyl ethers (e.g., polyoxyethylene nonyl phenyl ether, etc.), macrogols, polyoxyethylene polyoxypropylene glycols (e.g., poloxamer 407, poloxamer 235, poloxamer 188, poloxamine, etc.), etc.); (3) Cationic surfactants (e.g., benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, hexadecyltrimethylammonium bromide, dequalinium chloride, etc.); (4) Amphoteric surfactants (e.g., cocamidopropyl betaine, cocamidopropyl hydroxysultaine, etc.).

[0035] As the gas to be enclosed in the microbubbles or nanobubbles in the production method of the present invention, any gas that can be used for producing bubbles can be used. For example, the gas may be one or a mixture of two or more selected from perfluorohydrocarbons (e.g., perfluoropropane (C3F8), perfluorobutane, etc.), air, nitrogen, ozone, oxygen, argon, carbon dioxide, propane (C3H8), carbon monoxide (CO), and helium, but is not limited thereto. Preferred are perfluorohydrocarbons (e.g., perfluoropropane, perfluorobutane, etc.), air, nitrogen, ozone, oxygen, and argon. More preferred are perfluorohydrocarbons (e.g., perfluoropropane, perfluorobutane, etc.), air, etc. When air is used as the gas, nanobubbles can be produced inexpensively and easily. More preferably, the gas is perfluoropropane or perfluorobutane.

[0036] The mixture containing microbubbles and nanobubbles before freeze-drying is usually in a liquid state. In other words, the microbubbles and nanobubbles before freeze-drying are usually present in a state of being contained in a liquid. In the production method of the present invention, the liquid containing microbubbles and nanobubbles is not particularly limited, and may be, for example, tap water, deionized water, distilled water, sterile distilled water, purified water for injection, or ultrapure water. In addition, the liquid may contain other components in addition to the microbubbles and nanobubbles.

[0037] Methods for producing a mixture of microbubbles and nanobubbles are known per se. Common methods include a method of crushing gas by high-speed swirling (or high-speed vibration) to generate a mixture of microbubbles and nanobubbles (high-speed swirling liquid flow method), and a method of pressurizing gas and dissolving it in supersaturation and rapidly depressurizing the liquid to precipitate microbubbles and nanobubbles (pressure dissolution method). Many devices for generating microbubbles and nanobubbles are commercially available, and these may be used. Examples of commercially available devices include, but are not limited to, OM4-MD5-045 manufactured by Auratec Co., Ltd., Microbubble Generator manufactured by Nikuni Co., Ltd., YJ manufactured by By Clean Co., Ltd., Microbubble Generator manufactured by Aquaair Co., Ltd., and Microblade manufactured by Royal Electric Co., Ltd. As an example, a method for preparing a mixture of microbubbles and nanobubbles having a human serum albumin shell by high-speed vibration will be specifically described below, but the method for producing a mixture of microbubbles and nanobubbles having an albumin shell is not limited to this.

[0038] (1) Inject perfluoropropane gas (C3F8) into an empty glass vial and seal it with a rubber stopper or similar. (2) Using a syringe or other tool, additional perfluoropropane gas and an aqueous solution of human serum albumin are added through the rubber stopper. (3) Vibrate the vial at high speed. (4) After shaking, centrifuge the vial and cool it on ice. (5) The process of vibration, centrifugation, and cooling is repeated multiple times to generate bubbles.

[0039] The lower limit of the concentration of human serum albumin in the aqueous human serum albumin solution used in the above (2) is usually 0.001% or more, preferably 0.005% or more, 0.01% or more, 0.02% or more, 0.03% or more, or 0.04% or more, and more preferably 0.05% or more. The upper limit of the concentration of human serum albumin is usually 0.9% or less, preferably 0.85% or less, 0.8% or less, 0.75% or less, 0.7% or less, or 0.65% or less, and more preferably 0.6% or less. If the concentration of human serum albumin in the solution is 1% or more, the viscosity of the solution increases, and it may become difficult to generate nanobubbles by high-speed vibration. In one aspect, the concentration of human serum albumin in the human serum albumin solution is 0.001 to 0.9%, preferably 0.005 to 0.85%, 0.01 to 0.8%, 0.02 to 0.75%, 0.03 to 0.7%, or 0.04 to 0.65%, and more preferably 0.05 to 0.6%.

[0040] In the past, differences in the buoyancy characteristics of microbubbles and nanobubbles were used to separate nanobubbles from a mixture of microbubbles and nanobubbles. Microbubbles have buoyancy, while nanobubbles do not. Therefore, by leaving a liquid containing microbubbles to stand or centrifuging it, the microbubbles rise to the surface and the nanobubbles remain in the liquid, allowing the two to be separated. However, in such previous methods that utilize the buoyancy of microbubbles, microbubbles remain in the nanobubble fraction, making it a major challenge to prepare nanobubbles of high purity.

[0041] The production method of the present invention is characterized by freeze-drying a mixture (or a mixed liquid) of microbubbles and nanobubbles. By freeze-drying the mixture of microbubbles and nanobubbles, it is possible to collapse substantially all of the microbubbles while maintaining the nanobubbles.

[0042] The freeze-drying of the mixture of microbubbles and nanobubbles may be carried out using a method known per se. In general, the mixture can be freeze-dried by, for example, freezing the mixture using liquid nitrogen or the like and then vacuum drying it. The temperature and time used for freezing the mixture are not particularly limited, and any temperature and time may be used as long as the mixture of microbubbles and nanobubbles is sufficiently frozen. In addition, the pressure and time used for drying the frozen product are also not particularly limited, and any pressure and time may be used as long as the mixture of frozen microbubbles and nanobubbles can be sufficiently dried. In a preferred embodiment of the present invention, the freeze-drying of the mixture of microbubbles and nanobubbles may be vacuum freeze-drying. The vacuum freeze-drying may also be carried out using a method known per se.

[0043] As described above, the manufacturing method of the present invention is characterized in that the difference in buoyancy characteristics between microbubbles and nanobubbles is not utilized in the step of separating nanobubbles from a mixture of microbubbles and nanobubbles. Thus, in one aspect of the manufacturing method of the present invention, the manufacturing method of the present invention is characterized in that it does not include a step of subjecting the mixture of microbubbles and nanobubbles to centrifugation.

[0044] The nanobubbles produced by the production method of the present invention are characterized by being substantially free of microbubbles as contaminants. In other words, the remaining rate of microbubbles as contaminants when nanobubbles are produced by the production method of the present invention is calculated by the following formula:

[0045] Microbubble survival rate (%) = (microbubble concentration after freeze-drying / microbubble concentration before freeze-drying) x 100

[0046] When defined as above, the residual rate of microbubbles may be usually 1% or less, preferably 0.8% or less, 0.6% or less, or 0.4% or less, more preferably 0.3% or less, and particularly preferably 0.1% or less. In one embodiment, the residual rate of microbubbles may be usually 0 to 1%, preferably 0 to 0.8%, 0 to 0.6%, 0 to 0.4%, or 0 to 0.3%, and particularly preferably 0 to 0.1%.

[0047] In other words, the reduction in the residual rate of microbubbles can be regarded as the removal of the microbubbles. From this viewpoint, the removal rate of the microbubbles can be defined by the following formula:

[0048] Microbubble removal rate (%) = 100 - (microbubble remaining rate)

[0049] The microbubble removal rate can be usually 99% or more, preferably 99.2% or more, 99.4% or more, or 99.6% or more, more preferably 99.7% or more, and particularly preferably 99.9% or more. In one embodiment, the microbubble removal rate can be usually 99 to 100%, preferably 99.2 to 100%, 99.4 to 100%, 99.6 to 100%, or 99.7 to 100%, and particularly preferably 99.9 to 100%.

[0050] The hollow diameter of the nanobubbles produced by the production method of the present invention can be usually 1 nm or more, preferably 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more, more preferably 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 95 nm or more, and particularly preferably 100 nm or more. The upper limit of the hollow diameter of the nanobubbles can be usually less than 1000 nm, preferably 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, or 500 nm or less, more preferably 400 nm or less, 350 nm or less, 330 nm or less, 320 nm or less, or 310 nm or less, and particularly preferably 300 nm or less. In one embodiment, the hollow diameter of the nanobubbles produced by the production method of the present invention can be, but is not limited to, usually 1 to less than 1,000 nm, preferably 10 to 900 nm or less, 20 to 800 nm or less, 30 to 700 nm or less, 40 to 600 nm or less, 50 to 500 nm or less, more preferably 60 to 400 nm or less, 70 to 350 nm or less, 80 to 330 nm or less, 90 to 320 nm or less, 95 to 310 nm or less, and particularly preferably 100 to 300 nm or less.

[0051] In one embodiment, the nanobubbles produced by the production method of the present invention can satisfy the following conditions in terms of hollow diameter and residual rate of microbubbles: [1] Hollow diameter: 1 to less than 1000 nm, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [2] Hollow diameter: 10 to 900 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [3] Hollow diameter: 20 to 800 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [4] Hollow diameter: 30 to 700 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [5] Hollow diameter: 40 to 600 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [6] Hollow diameter: 50 to 500 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [7] Hollow diameter: 60 to 400 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [8] Hollow diameter: 70 to 350 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0% [9] Hollow diameter: 80 to 330 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0%

[10] Hollow diameter: 90 to 320 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0%

[11] Hollow diameter: 95 to 310 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0%

[12] Hollow diameter: 100 to 300 nm or less, and Residual rate: 0-1%, 0-0.8%, 0-0.6%, 0-0.4%, 0-0.3%, 0-0.1%, or 0%

[0052] The nanobubbles produced by the present invention can be stored for a long period in a freeze-dried state. Previously, nanobubbles having a shell made of peptides or proteins composed of amino acids (e.g., nanobubbles having an albumin shell) were thought to be difficult to freeze-dry, and therefore no products that could be stored for years had been developed. However, the present invention makes it possible to prepare nanobubbles having a shell made of peptides or proteins composed of amino acids in a state where microbubbles are less contaminated and can be stored for a long period of time.

[0053] The nanobubbles produced by the present invention can be easily regenerated from the freeze-dried state by reconstituting them with distilled water, etc. As demonstrated in the following examples, nanobubbles regenerated after freeze-drying maintain the same functions as the nanobubbles before freeze-drying.

[0054] Furthermore, when regenerating nanobubbles prepared by freeze-drying, a nanobubble solution having a desired nanobubble concentration can be produced by appropriately adjusting the amount of liquid (e.g., distilled water) in which the nanobubbles are dissolved. Conventionally, it took a relatively long time and high cost to produce a high-concentration nanobubble solution that is substantially free of microbubbles as impurities, but according to the present invention, a high-concentration nanobubble solution can be easily produced by essentially performing a freeze-drying process. Focusing on this aspect, the present invention also provides a "method for adjusting the concentration of nanobubble water, comprising the steps of freeze-drying a mixture of nanobubbles and microbubbles having a shell made of a peptide or protein, and immersing the nanobubbles obtained by freeze-drying in a regenerating solution."

[0055] In addition, in view of the aspect of the present invention in which the purity of the nanobubbles is increased by freeze-drying a mixture of microbubbles and nanobubbles having a shell made of a peptide or protein, thereby collapsing only the microbubbles, the present invention can be rephrased as "a method for removing microbubbles from a mixture of nanobubbles and microbubbles having a shell made of a peptide or protein (hereinafter, may be referred to as "the removal method of the present invention")".

[0056] The removal rate of microbubbles in the removal method of the present invention is the same as that explained in the manufacturing method of the present invention.

[0057] Alternatively, when a mixture of microbubbles and nanobubbles is subjected to freeze-drying, the microbubbles collapse and relatively large nanobubbles also collapse partially, resulting in the average particle size (average hollow diameter of nanobubbles) of the nanobubbles produced by the production method of the present invention being smaller than the average particle size of the nanobubbles before freeze-drying. Focusing on this aspect of the present invention, the present invention can also be interpreted as a "method for micronizing nanobubbles (hereinafter, sometimes referred to as the "micronization method of the present invention")".

[0058] In the micronization method of the present invention, the average particle size of the nanobubbles is usually 1 nm or more, preferably 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more. Above, 210nm or more, 220nm or more, 230nm or more, 240nm or more, 250nm or more, 260nm or more, 270nm or more, 280nm or more, 290nm or more, 300nm or more, 310nm or more, 320nm 330nm or more, 340nm or more, 350nm or more, 360nm or more, 370nm or more, 380nm or more, 390nm or more, 400nm or more, 410nm or more, 420nm or more, 430nm or more, 440n m or more, 450nm or more, 460nm or more, 470nm or more, 480nm or more, 490nm or more, 500nm or more, 510nm or more, 520nm or more, 530nm or more, 540nm or more, 550nm or more, 56 0nm or more, 570nm or more, 580nm or more, 590nm or more, 600nm or more, 610nm or more, 620nm or more, 630nm or more, 640nm or more, 650nm or more, 660nm or more, 670nm or more, 6 It may be 80nm or more, 690nm or more, 700nm or more, 710nm or more, 720nm or more, 730nm or more, 740nm or more, 750nm or more, 760nm or more, 770nm or more, 780nm or more, 790nm or more, 800nm ​​or more, 810nm or more, 820nm or more, 830nm or more, 840nm or more, 850nm or more, 860nm or more, 870nm or more, 880nm or more, 890nm or more, or 900nm or more.The upper limit of the average particle size of the nanobubbles is usually less than 1000 nm, preferably 990 nm or less, 980 nm or less, 970 nm or less, 960 nm or less, 950 nm or less, 940 nm or less, 930 nm or less, 920 nm or less, 910 nm or less, 900 nm or less, 890 nm or less, 880 nm or less, 870 nm or less, 860 nm or less, 850 nm or less, 840 nm or less, 830 nm or less, 820 nm or less, 810 nm or less, 800 nm or less, nm or less, 790nm or less, 780nm or less, 770nm or less, 760nm or less, 750nm or less, 740nm or less, 730nm or less, 720nm or less, 710nm or less, 700nm or less, 690nm or less, 68 0nm or less, 670nm or less, 660nm or less, 650nm or less, 640nm or less, 630nm or less, 620nm or less, 610nm or less, 600nm or less, 590nm or less, 580nm or less, 570nm or less, 56 0nm or less, 550nm or less, 540nm or less, 530nm or less, 520nm or less, 510nm or less, 500nm or less, 490nm or less, 480nm or less, 470nm or less, 460nm or less, 450nm or less, 4 40nm or less, 430nm or less, 420nm or less, 410nm or less, 400nm or less, 390nm or less, 380nm or less, 370nm or less, 360nm or less, 350nm or less, 340nm or less, 330nm or less, It can be 320nm or less, 310nm or less, 300nm or less, 290nm or less, 280nm or less, 270nm or less, 260nm or less, 250nm or less, 240nm or less, 230nm or less, 220nm or less, 210nm or less, 200nm or less, 190nm or less, 180nm or less, 170nm or less, 160nm or less, 150nm or less, 140nm or less, 130nm or less, 120nm or less, 110nm or less, or 100nm or less. In one embodiment, the average particle size of the nanobubbles obtained by the micronization method of the present invention can be, but is not limited to, usually 1 to less than 1,000 nm, preferably 10 to 900 nm or less, 10 to 800 nm or less, 10 to 700 nm or less, 10 to 600 nm or less, or 10 to 500 nm or less, more preferably 10 to 400 nm or less, 10 to 350 nm or less, 50 to 350 nm or less, 100 to 350 nm or less, 150 to 350 nm or less, or 150 to 300 nm or less.

[0059] 2. Dry nanobubbles or composition containing same The present invention also provides dry nanobubbles having a shell made of a peptide or protein, or a composition containing the same (hereinafter sometimes referred to as "nanobubbles of the present invention" or "composition of the present invention").

[0060] The nanobubbles of the present invention are nanobubbles themselves (i.e., freeze-dried nanobubbles) having a shell made of a peptide or protein produced by the production method of the present invention. The composition of the present invention is obtained by immersing the nanobubbles of the present invention in an appropriate solution to regenerate the dried nanobubbles. The terms such as purity and average particle size of the nanobubbles are the same as those explained in the production method of the present invention and the microparticulation method of the present invention.

[0061] The composition of the present invention may contain components other than the nanobubbles of the present invention, so long as the desired effects of the present invention are achieved.

[0062] The nanobubbles of the present invention and the composition of the present invention can be used not only for the purpose of enhancing the drug function in DDS, but also for other purposes. For example, a method of suppressing the growth of microorganisms using microbubbles or nanobubbles has been reported, and is used in the fields of pharmaceutical and food manufacturing as well as for improving the water quality of lakes and ponds. As demonstrated in the following examples, the composition of the present invention has a biofilm formation suppressing effect, and can therefore be used for such purposes.

[0063] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0064] [material and method]

[0065] 1. Preparation of a mixture of albumin-shelled microbubbles and nanobubbles A mixture of albumin-shelled microbubbles and nanobubbles was prepared as follows. 5 mL of perfluoropropane gas (C3F8, Takachiho Chemical Industry Co., Ltd.) was injected into an empty glass vial. An additional 1.5 mL of perfluoropropane gas and 3.6 mL of 0.06% human serum albumin (Albuminar 25, CSL Behring LLC) in distilled water were filled into the vial, closed with a rubber stopper and sealed with an aluminum cap, through the rubber stopper using a 23-gauge needle. The vial was then placed into a high-speed vibrating tissue homogenizer device (Precellys Evolution, Bertin Instruments) and vibrated at 6000 rpm for 80 seconds. After shaking, the vial was centrifuged at 100G for 2 minutes (MX-401, TOMY) and cooled on ice for 3 minutes. The shaking, centrifugation, and cooling were repeated three times, with one additional shaking phase at the end. An 18-gauge needle was inserted into the rubber stopper of the vial to release the pressure. The temperature change of the aqueous human serum albumin solution due to vibration and cooling was measured over time using a digital thermometer (TX1003, Yokogawa Measurement Co., Ltd.).

[0066] The aqueous solution containing microbubbles and nanobubbles was immersed in liquid nitrogen and rapidly frozen in a vial with a perforated rubber stopper. The frozen material was freeze-dried for 48 hours in a freeze dryer (Free Zone 4.5, Labconco Corporation) at a vacuum pressure of 20 Pa and a collector temperature of -50°C.

[0067] The vials containing the lyophilized nanobubbles were reclosed with rubber stoppers, sealed with aluminum caps to prevent moisture absorption, and stored at 4°C.

[0068] When not freeze-dried (i.e., control), the suspension containing the bubbles was mixed homogeneously by pipetting, stored at 4°C within 2 hours, and then subjected to measurement.

[0069] 2. Morphological Observation of Freeze-Dried Nanobubbles Using a Scanning Electron Microscope Nanobubble lyophilisates and controls were carefully torn off with tweezers and mounted with the cross-section facing up on an aluminium stub with carbon tape. The lyophilisate pieces were coated with a layer of osmium using an OPC-80 osmium plasma coater (Nippon Laser & Electronics Lab). Samples were observed using a scanning electron microscope (SEM) (JSM-7500F, JEOL) at 5 kV. Areas of the lyophilisates were randomly selected and observed at magnifications ranging from 1000× to 30000×.

[0070] 3. Evaluation of physical properties and survival of regenerated nanobubbles The freeze-dried material containing nanobubbles was dissolved in 3.6 mL of distilled water in the same manner as before freeze-drying. The physical properties of the nanobubbles were evaluated by analyzing the sample using nanoparticle tracking analysis, flow cytometry analysis, and resonance mass measurement.

[0071] [Nanoparticle tracking analysis] The nanobubble particle size was measured by a nanoparticle tracking analysis (NTA) device (NanoSight LM10, Malvern Instruments). The nanoparticle suspension was irradiated with a red laser with a wavelength of 638 nm. The nanoparticle movement was visualized by light scattering and Brownian motion using a CCD camera (C11440-50B, Hamamatsu Photonics K.K.). This system automatically detects the center position of the nanoparticle and tracks the movement of each particle in a two-dimensional plane to calculate the average movement distance under Brownian motion. Images of particle movement in the NTA were recorded for 60 seconds at room temperature. The range of particle size measurement by the NTA method was adjusted to 10 nm to 1000 nm. The particle size was evaluated by the average movement distance according to the Stokes-Einstein equation. 0.5 mL of the nanobubble suspension was injected into the sample measurement chamber of the NanoSight system with a 1.0 mL plastic syringe (Terumo). Sample image capturing and data analysis were performed using measurement application software (NTA 3.2 Dev Build3.2.16). All sample measurements were performed independently for each sample. Particle sizes are presented as the mean and mode ± standard error of the mean of triplicate measurements. Nanobubble size after reconstitution was compared to non-lyophilized, centrifuged samples.

[0072] [Flow cytometry analysis] The size ratio and number of nanobubbles were measured using a flow cytometer (CytoFLEX, Beckman Coulter). The flow cytometer was equipped with a 405 nm (violet) laser for nanoparticle detection, and was set up to measure side scatter (SS) from the violet laser (violet SS) for enhanced nanoparticle detection. The violet SS signal resolution limit for particle detection was 200 nm. Better resolution can be obtained with SS than with forward scatter (FS) signals, which is suitable for measuring small particles (e.g., nanoscale particles). Violet-SS To relate the violet side scatter area (SS-A) to particle size, the flow cytometer was calibrated with beads of known size (Wisgrill et al., Cytometry A. 2016 Jul;89(7):663-72.; Zucker et al., Cytometry A. 2016 Feb;89(2):169-83.). Polystyrene standard beads (500 nm, qNano Calibration Particle, Izon Science; 1000 nm, Archimedes Standard polystyrene beads, Malvern Instruments) were suspended in ultrapure water and measured in advance with a flow cytometer. The obtained violet SS-A signal of nanobubbles was then analyzed by CytExpert software version 2.0 (Beckman Coulter, Inc.). Before the experiment, gates of violet SSC-A values ​​were created based on the size of each standard bead in the range of 500 to 1000 nm to determine the size of nanobubbles. Using these data, the number of nanobubbles present in the signal band of each size was measured. The number of nanobubbles was diluted 10 times before measurement, and the concentration of the stock suspension was retrospectively back-calculated. Based on the concentration of nanobubbles in the aqueous solution after centrifugation without lyophilization, the total number and the percentage remaining after lyophilization-dissolution for each size range were calculated.The undiluted solution was measured directly, and particles exceeding the gate of the Violet SSC-A value of 1000 nm standard beads were counted as microbubbles to calculate the microbubble concentration. The residual rate and removal rate were calculated from the microbubble concentration immediately after preparation of the microbubble / nanobubble mixed solution and after freeze-drying and dissolution.

[0073] [Resonance mass measurement] To confirm that the nanoparticles present after freeze-drying-dissolving are indeed buoyant bubbles, the particle mass was measured by a resonance mass measurement (RMM) system (Archimedes, Malvern Instruments Ltd) based on our previous report (Watanabe et al., Heliyon 2019, 5, e01907.). The RMM is used so that the sample solution passes through a microfluidic flow channel in the cantilever. Particles passing through the microfluidic flow channel were detected based on the instantaneous shift in the resonance frequency of the cantilever, which is related to the mass change caused by the passage of particles with a density different from that of the solution. According to the direction of the frequency shift, positively buoyant particles and negatively buoyant particles can be clearly distinguished (Patel, AR et al., Analytical chemistry 2012, 84, 6833-6840., Burg, TP et al., Nature 2007, 446, 1066-1069.). In this example, 2 × 2 μm 2The resonator in Archimedes Hi-Q nano sensor (Malvern Instruments Ltd) with internal microfluidic flow channel dimensions of 0.01 Hz was used. For all measurements, the directional limit or threshold of 0.01 Hz was manually selected based on the baseline noise observed in the control sample PBS solution. The nanobubble suspension was fed into the Hi-Q nanosensor and the measurement continued for 20 min at room temperature. The buoyant mass was calculated from the temporal resonant frequency shift by using Particle Lab Software version 1.9.81 (Malvern Instruments Ltd).

[0074] 4. Ultrasound (US) responsiveness of regenerated nanobubbles To confirm that the nanoparticles present after freeze-drying and dissolution are indeed US-responsive bubbles, the size distribution of the nanoparticles was measured before and after sonication. Sonication was based on our previous report (Kida, H. et al., Frontiers in Pharmacology 2022, 13.). Briefly, the nanobubble suspension (100 μL) was placed in an acoustically transparent film-based 96-well multiwell cell culture plate (Sarstedt, Numbrecht). The culture plate was fixed onto the surface of the US transducer via an acoustically transparent gel (Aquasonic 100 gel, Parker lab). US was irradiated by a sonoporator (SP100, Sonidel Limited) with a transducer (diameter 1.6 cm), a driving frequency of 1 MHz, a burst rate of 100 Hz, and a 50% duty ratio (Figure 1A). The method of US irradiation is similar to that of the microscale in vitro sonoporation system using a 96-well multiwell plate containing cultured cells described below. The diameter of the nanobubbles was determined by the different intensities (0, 1, 2, or 5 W / cm 2 The changes in the number and distribution of nanobubbles before and after US irradiation were measured using the nanoparticle tracking analysis, flow cytometry analysis, and resonance mass spectrometry described above.

[0075] 5. In Vitro Ultrasound Image Characterization of Regenerated Nanobubbles US imaging was performed to evaluate the functionality of the regenerated nanobubbles as a US contrast agent. A flow phantom (7 cm × 8 cm × 11 cm) (Figure 2) made of a US gel pad (Aquaflex ultrasound gel pad; Parker lab) was custom-made for the US contrast-enhanced imaging experimental setup. Two parallel flow tubes with a diameter of 5 mm for flowing nanobubble aqueous solutions were fabricated at a depth of 1.5 cm from the surface in the horizontal long axis direction of the phantom and 2 cm apart. The regenerated nanobubble solution or the control was diluted 3-fold with distilled water, filled into a syringe, and injected directly into the flow tube in the same direction at a flow rate of 2 mL / min through a 3.1 mm inner diameter polyvinyl chloride tube (SF0ET2022L, Terumo) connected to the hole of the flow tube by an autoinjector (YSP-201, Terumo). Acoustic evaluation of the regenerated nanobubbles was performed by a diagnostic US imaging system (LOGIQ E9, GE Healthcare) with a broad-spectrum intraoperative linear array L8-18i-D probe (4–14 MHz). The US probe was placed and fixed on the upper surface of the flow phantom. US B-mode images of the US flow phantom were acquired with and without the use of Coded Harmonic Angio (CHA, mechanical index 0.6) or Amplitude Modulation (AM, mechanical index 0.28) contrast modes.

[0076] 6. Luciferase mRNA Transfection and Evaluation of Expression In Vitro [mRNA transfection] In order to confirm the effect of intracellular delivery of mRNA by US responsiveness of regenerated nanobubbles, an experiment was conducted based on the previous report by the present inventors (Kida, H. et al., Frontiers in Pharmacology 2022, 13.). The freeze-dried nanobubble-containing material was dissolved in opti-MEM in an amount equal to the aqueous solution before freeze-drying. mRNA encoding Gaussia luciferase (GLuc) was added to the nanobubbles or control solution at a final concentration of 10 μg / mL, respectively. A schematic diagram of all steps of this experiment is shown in Figure 1. Each oral squamous cell carcinoma cell (HSC-2) culture medium in a 96-well plate with an acoustically transparent bottom was replaced with 50 μL of regenerated nanobubble medium containing 500 ng of mRNA (Figures 1B and C). US (SP100, Sonidel Limited) was irradiated to the bottom of the medium plate containing HSC-2 cells, nanobubbles and genes (Figure 1D). After the US irradiation treatment, the solution containing nanobubbles was removed. Then, 100 μL of culture medium was refilled into each culture well and incubated in a humidified 5% CO 2 The cells were incubated at 37° C. in an ambient atmosphere (FIG. 1E). After 24 hours, luciferase expression assays and cell viability assays were performed (FIGS. 1F and G).

[0077] [Luciferase expression assay] In vitro luciferase activity was determined using a Spark® multimode microplate reader (Tecan, Mannedorf). After 24 h incubation following cell sonication, 10 μL of culture supernatant was collected from each incubation well of a Costar 96 well white solid plate (Corning). 100 μL / 100 μL of coelenterazine (Gold Biotechnology) solution dissolved in 0.01% Tween® 20 / 0.1 mM EDTA / PBS was added to each well. Relative luminescence unit (RLU) values ​​from 2 to 12 seconds after addition were plotted and summed.

[0078] [Cell viability assay] To determine the number of viable cells in the cytotoxicity assay (CellTiter 96 AQue-ous One Solution Cell Proliferation Assay system (Promega)), the number of viable HSC-2 cells was measured by colorimetry using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). 20 μL of Cell Titer Solution Reagent was added to each well from which a portion of the supernatant was removed for luciferase assay. After 2 h of incubation, the absorbance was recorded at 490 nm using a 96-well plate reader Multiskan™ Go (Thermo Fisher Scientific). The viability of treated cells was calculated as the ratio of the number of viable cells to the number of untreated viable cells in the control.

[0079] 7.Statistical analysis Measurement data were expressed as mean ± standard error of the mean (sem). Data were analyzed using unpaired t-test with Welch's correction. Statistically significant differences between groups were analyzed using Microsoft Excel (Version 2212, Microsoft). A probability value of p < 0.05 was considered statistically significant.

[0080] [Example 1] The shell of human serum albumin-based nanobubbles is retained upon freeze-drying. HSA solutions bubbled at a maximum temperature of 25°C for four vibration cycles were flash frozen and vacuum dried (Fig. 9). The resulting freeze-dried products, whether or not bubbles were present, were cotton-like to the naked eye (Fig. 3). These structures were soft and easily torn apart when pinched with tweezers. The two could not be distinguished by visual inspection of hardness with tweezers or by visual observation.

[0081] Low-magnification SEM (×1,000) of lyophilized material prepared from the bubble-containing solution revealed numerous dried bubble shells with diameters exceeding 1 μm. Shell pores were observed in the majority of these microbubbles (Fig. 4A). High-magnification SEM (×10,000) showed spherical objects that appeared to be nanobubbles (Fig. 4B). Ultra-high-magnification SEM (×30,000) confirmed spherical HSA-nanobubbles with pores in their shells, although it was difficult to completely distinguish them from the irregular, tangled-shaped albumin aggregates present among the HSA-nanobubbles (Fig. 4C). On the other hand, low- and ultra-high-magnification SEM (×1,000 to ×30,000) observations of lyophilized material from the non-bubbled solution revealed no structures resembling microbubbles or nanobubbles, and only irregularly shaped albumin aggregates (Fig. 4D-F).

[0082] [Example 2] Nanobubbles are regenerated by dissolving a freeze-dried material containing bubbles of human serum albumin shell. The freeze-dried material containing HSA nanobubbles was redissolved in the same amount of distilled water as before freeze-drying. The particle concentration and particle distribution before and after freeze-drying were back-calculated from the measurements at 10-fold dilution by FCM (Figure 5A, Table 1). The concentration of HSA nanobubbles in the solution before vacuum freeze-drying was 1.2 × 10 9 / mL. In comparison, the bubble concentration in the regenerated nanobubble solution was 7.8 × 10 8 / mL. In other words, it was found that 64.5% of the nanobubbles were retained by the freeze-drying procedure. Nanobubbles with a diameter of less than 200 nm accounted for 102.0% (3.0 × 10 8 / mL to 3.1×10 8 / mL). Nanobubbles with diameters of 200 to 500 nm and nanobubbles with diameters of more than 500 nm accounted for 57.3% (7.9 × 10 8 / mL to 4.5×10 8 / mL) and 17.7% (1.2 × 10 8 / mL to 2.2×10 7 The nanobubble concentration was measured by NTA and was 13.8×10 9 / mL to 4.8 × 10 after lyophilization 9 / mL (34.8%) (Figure 5B). The average nano-bubble size was 266.7 ± 17.1 nm before vacuum freeze-drying, but decreased to 224.8 ± 13.8 nm after vacuum freeze-drying. In the RMM measurement, the nanobubble concentration was 11.5 × 10 7 / mL to 9.6 × 10 after lyophilization 7 / mL (83.2%) (Figure 5C). 100.0% of the nanobubbles present before vacuum freeze-drying were positively buoyant, with an average floating mass of -2.6 fg. On the other hand, 97.7% of the nanobubbles present after vacuum freeze-drying were positively buoyant, with an average floating mass of -2.1 fg.

[0083] [Table 1]

[0084] (Table 1: Change in nanobubble concentration due to freeze-drying. The values ​​in parentheses indicate the percentage maintained from the concentration before freeze-drying. NBs: nanobubbles.)

[0085] [Example 3] Freeze-drying can efficiently remove microbubbles from a mixture of microbubbles and nanobubbles. The microbubble concentrations in the microbubble / nanobubble mixture immediately after preparation and in a solution obtained by redissolving the lyophilized material in an equal amount of pure water were compared using FCM measurements. The results are shown in Figure 10. The microbubble concentration in the solution before lyophilization was 1.4×10 7 The concentration of microbubbles in the solution redissolved in an equal volume of pure water after freeze-drying was 5.6 × 10 4 The residual rate of microbubbles before and after freeze-drying was 0.41%, and the removal rate was 99.59%.

[0086] [Example 4] Nanobubbles can be regenerated from the freeze-dried material by concentrating or diluting it to any desired concentration. The freeze-dried material was dissolved in pure water in an equal amount and one-third the amount of the solution before drying, and the concentration of the regenerated nanobubbles was compared using FCM measurement. The results are shown in Figure 11. The nanobubble concentration when dissolved in the same amount of pure water was 6.9x10 8 / mL. In contrast, the nanobubble concentration when dissolved in 1 / 3 the amount of pure water was 1.2x10 9 / mL, which was 1.7-fold higher than the concentration obtained when reconstituted with an equal volume of pure water.

[0087] [Example 5] Nanobubbles regenerated from a freeze-dried state have the effect of inhibiting biofilm formation. The inhibitory effect on bacterial biofilm formation was investigated using concentrated nanobubbles obtained by regenerating freeze-dried nanobubbles and an albumin solution of the same concentration (n=6).

[0088] The freeze-dried nanobubbles were dissolved in MHB medium in a volume of 1 / 3 of the volume of the solution before drying to regenerate the nanobubbles. 8 The mixture was diluted to CFU / mL, added to a 96 multi-well plate at 180 μL / well, and a pin plate from Biofilm Formation Assay Kit (Dojindo Laboratories) was attached. 24 hours after the start of culture at 37°C, the medium was replaced with fresh MHB medium containing nanobubbles, and cultured for another 72 hours. After the culture was completed, the biofilm formed on the pin plate was stained with 0.1% Crystal Violet solution. The pin plate was washed with saline and then immersed in a new 96 multi-well plate filled with ethanol to extract Crystal Violet. The absorbance at 590 nm of the ethanol from which Crystal Violet was extracted was measured. The amount of biofilm formed calculated from the absorbance under the condition of culture using MHB medium containing the same concentration of albumin was set to 100%, and the biofilm formation rate under the condition of culture using HMB medium containing nanobubbles was calculated. The results are shown in Figure 12. When bacteria were cultured using a medium containing nanobubbles, the biofilm formation rate was suppressed by 21.6%.

[0089] [Example 6] Nanobubbles regenerated from freeze-dried materials collapse when irradiated with ultrasound. The nanobubble solution regenerated from the freeze-dried material was irradiated with ultrasound at 1, 2 or 5 W / cm. 2US irradiation at 1000 nm increased the nanobubble concentration to 6.3×10 in FCM measurements. 8 / mL to 2.1 × 10 8 / mL (33.3%), 1.6 × 10 8 / mL (24.7%) or 9.9 × 10 7 / mL (15.7%) (Figure 6A, Table 2). 2 For nanobubbles with a diameter of less than 200 nm, the US irradiation of 8 / mL to 7.1 × 10 7 / mL (34.7%), and for nanobubbles with diameters of 200 to 500 nm, 4.0 × 10 8 / mL to 2.5 × 10 7 / mL (6.2%), and for nanobubbles with a diameter of more than 500 nm, 2.2 × 10 7 / mL to 2.5 × 10 6 / mL (11.3%), respectively. In the NTA measurements, the concentration was reduced to 1, 2 or 5 W / cm 2 The US irradiation was 35.3 × 10 8 / mL nanobubble concentration, respectively, 18.1 × 10 8 / mL (51.3%), 7.1 × 10 8 / mL (20.0%) and 5.2 × 10 8 / mL (14.8%) (Figure 6B). The average nanobubble size decreased from 266.1 ± 6.7 nm to 189.3 ± 11.1 nm, 192.5 ± 3.6 nm, and 166.8 ± 2.2 nm, respectively. RMM measurements showed that the nanobubble concentration was 95.7 × 10 6 / mL to 26.0 × 10 6 / mL (27.2%), 4.4 × 10 6 / mL (4.6%) and 5.49 × 10 6 / mL (5.7%). The mean buoyant mass of nanobubbles was -2.1 fg before sonication and -1.9 fg, -0.3 fg, and -1.25 after sonication, respectively. Positively buoyant particles were 97.7% before sonication and 98.4%, 76.5%, and 100.0% after sonication, respectively (Figure 6C).

[0090] [Table 2]

[0091] (Table 2: Change in regenerated nanobubble concentration due to sonication. Values ​​in parentheses indicate the percentage maintained from the concentration before sonication. NBs: nanobubbles.)

[0092] [Example 7] Nanobubbles regenerated from lyophilized material are echogenic. Diluted solutions containing or not containing nanobubbles regenerated from the lyophilized material were perfused through the tubes of a flow phantom, and their echogenicity was observed. In the case of B-mode imaging alone, no change in echogenicity was detectable in the tubes for either solutions containing or not containing nanobubbles (Fig. 7A). An increase in brightness was detected only in the perfusion of nanobubble-containing solutions in CHA mode (Fig. 7B). On the other hand, in AM mode, no increase in brightness was observed in the tubes for either solutions containing or not containing nanobubbles (Fig. 7C).

[0093] [Example 8] Nanobubbles regenerated from freeze-dried material act as cavitation nuclei in mRNA sonoporation. In in vitro sonoporation, either with or without nanobubbles, mRNA transfection efficiency increased with a stepwise increase in acoustic intensity with US (1, 2 or 5 W / cm 2 ), but the increase in mRNA transfection efficiency by sonication was greater in the condition with nanobubbles. 2 Under the condition of US irradiation at the maximum acoustic intensity, the RLU value was 2.6 ± 0.1 (× 10 7 ) was reached. This value was 7.5 times higher than the value in the transfection condition where a solution without nanobubbles was used and US irradiation was performed at the same intensity (p=0.0002). 2 The cell viability after US irradiation at 37°C was 84.9% with nanobubbles and 90.7% without nanobubbles, respectively (p=0.6255) (Figure 8B). [Industrial Applicability]

[0094] According to the present invention, nanobubbles having a shell made of a highly safe biological protein such as albumin can be prepared with high purity. The nanobubbles can be used as a contrast agent and can also be used to deliver genes or drugs to cells. Therefore, the present invention is extremely useful, for example, in technical fields related to the diagnosis or treatment of diseases. Alternatively, since the nanobubbles prepared using the present invention have a biofilm formation inhibitory effect, they can also be applied to hygiene management in the production of food and pharmaceutical products and the improvement of environmental pollution by microorganisms.

Claims

1. A method for producing dry nanobubbles having a shell made of peptide or protein, comprising the step of freeze-drying a mixture of microbubbles having a shell made of peptide or protein and nanobubbles.

2. The manufacturing method according to claim 1, characterized in that the mixture of microbubbles and nanobubbles having shells made of peptides or proteins is not subjected to a separation step utilizing buoyancy.

3. The manufacturing method according to claim 1 or 2, wherein the freeze-drying is vacuum freeze-drying.

4. The method for producing a peptide or protein according to claim 1 or 2, wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme.

5. A method for removing microbubbles from a mixture of microbubbles having a peptide or protein shell and nanobubbles, comprising the step of freeze-drying the mixture of microbubbles having a peptide or protein shell and nanobubbles.

6. The method according to claim 5, characterized in that the mixture of microbubbles having a shell made of peptide or protein and nanobubbles is not subjected to a separation step utilizing buoyancy.

7. The method according to claim 5 or 6, wherein the freeze-drying is vacuum freeze-drying.

8. The method according to claim 5 or 6, wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme.

9. A method for miniaturizing nanobubbles having a shell made of peptide or protein, comprising the step of freeze-drying the nanobubbles having a shell made of peptide or protein.

10. The micronization method according to claim 9, wherein the freeze-drying is vacuum freeze-drying.

11. The micronization method according to claim 9 or 10, wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme.

12. Dry nanobubbles having a shell made of peptides or proteins, or a composition containing the same.

13. A composition according to claim 12, wherein the remaining percentage of microbubbles contained in the composition is 1% or less, and the average particle size of the nanobubbles is 1 to less than 1000 nm.

14. The nanobubble or composition according to claim 12 or 13, wherein the peptide or protein is selected from the group consisting of albumin, immunoglobulin G, and lysozyme.

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