阅读说明：本技术 一种包括用于递送药物的超声响应性微泡的脂质体的制备方法以及使用该方法的脂质体 (Method for preparing liposome comprising ultrasound-responsive microbubbles for delivering drugs and liposome using the same ) 是由 金哲右 朴东熙 元钟浩 于 2019-03-20 设计创作，主要内容包括：本发明提供一种包括用于递送药物的超声响应性微泡的脂质体的制备方法以及使用该方法的脂质体,其中,所述方法包括：(a)在产生内部包括惰性气体并在外面形成有第一壳体的超声响应F性微泡之后,通过挤出机均匀地形成所述超声响应性微泡的尺寸分布；以及(b)在产生包括内部尺寸分布均匀的所述超声响应性微泡和药物并在外面形成有第二壳体的脂质体后,通过挤出机均匀地形成所述脂质体的尺寸分布。(The present invention provides a method for preparing a liposome comprising ultrasound-responsive microbubbles for delivering a drug, and a liposome using the same, wherein the method comprises: (a) uniformly forming a size distribution of the ultrasound-responsive microbubbles by an extruder after generating the ultrasound-responsive F-type microbubbles including an inert gas inside and formed with a first shell outside; and (b) after liposomes comprising the ultrasound-responsive microbubbles and the drug having a uniform internal size distribution and formed with the second shell on the outside are produced, uniformly forming the size distribution of the liposomes by an extruder.)

1. A method of preparing liposomes comprising ultrasound-responsive microbubbles for delivering a drug, comprising:

(a) uniformly forming a size distribution of the ultrasound-responsive microbubbles by an extruder after generating the ultrasound-responsive microbubbles including the inert gas inside and formed with the first shell outside; and

(b) after liposomes comprising the ultrasound-responsive microbubbles and the drug having a uniform size distribution in the inside and formed with the second shell on the outside are produced, the size distribution of the liposomes is uniformly formed by an extruder.

2. The method of claim 1, further comprising:

(c) binding a targeting ligand reactive with a pathogen to which the drug is to be delivered to the second shell.

3. The method of claim 2, wherein:

in the step (c) of the present invention,

determining at least one of an antibody, protein, peptide and receptor reactive with the pathogen as the targeting ligand by performing ligand library screening against the pathogen, and binding the determined targeting ligand to the second shell.

4. The method of claim 3, wherein:

after introducing a carboxyl group (COOH) into the second shell, the carboxyl group is activated and then bound to the second shell by mixing the targeting ligand bound with an amino group (NH2) and reacting the carboxyl group with the amino group to form an amide.

5. The method of claim 4, wherein:

after introducing the carboxyl group into the second shell, adding EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and N-hydroxysuccinimide, (i) activating the carboxyl group, (ii) binding the targeting ligand to the second shell by mixing the targeting ligand bound to the amino group and reacting the carboxyl group with the amino group to form an amide.

6. The method of claim 1, wherein:

in the step (a),

dissolving a first mixture powder including a first lipid in a first solvent to produce a solution of a first shell material, mixing the solution of the first shell material with the inert gas, and then generating the ultrasound-responsive microbubbles by mechanical mixing.

7. The method of claim 6, wherein:

the first lipid comprises DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), HSPC (phosphatidylcholine), DDPC (1, 2-didecanoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-di (cis-13-di-erucyl) -sn-glycero-3-phosphorylcholine), DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine), DMPC (1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine), DLPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-dilauroyl-sn-glycero-3-phosphorylcholine), DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphorylcholine), egg PC (phosphorylcholine), DPPA (diphenylphosphoryl azide), DMPA-Na (1, 2-dimyristoyl-sn-glycero-3-phosphate), DPPA-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphate), DOPA-Na (1, 2-dioleoyl-sn-glycero-3-phosphate), DSPE (distearoylphosphatidylethanolamine), DMPE (dimyristoyl phosphatidylethanolamine), DOPE (dioleoyl phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE-glutaryl- (Na)2(1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine), eg PE (phosphatidylethanolamine), DSPG (distearoyl phosphatidylglycerol), DMPG-Na (1, 2-dimyristoyl-sn-glycerol-3-phosphoglycerol), DPPG-Na (1, 2-dipalmitoyl-sn-glycerol-3-phosphoglycerol), DOPG-Na (1, 2-dioleoyl-sn-glycerol-3-phosphoglycerol), DOPS (dimyristoyl phosphatidylserine), DMPS-Na (1, 2-dimyristoyl-sn-glycero-3-phosphoserine), DPPS-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphoserine), DOPS-Na (1, 2-dioleoyl-sn-glycero-3-phosphoserine), DSPS (distearoylphosphatidylserine), DSPE-mPEG (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-maleimide PEG-2000-Na, surfactant (b): at least one of Tween 80, Span 80, dipotassium glycyrrhizinate.

8. The method of claim 7, wherein:

the first lipid comprises the DPPC and the DPPA.

9. The method of claim 6, wherein:

the first mixture powder further includes at least one of albumin, a polymer, cholesterol, PEG, a surfactant, a protein, and a biodegradable polymer.

10. The method of claim 6, wherein:

the first solvent includes at least one of a salt solution or triple distilled water, glycerol, and propylene glycol.

11. The method of claim 6, wherein:

mixing the solution of the first shell material and the inert gas in a ratio of 1: 1 to 20: 1 by volume.

12. The method of claim 1, wherein:

in the step (a),

filtering the ultrasound-responsive microbubbles through a filter having a pore size of any one of 30nm to 1 μm and the extruder, uniformly forming a size distribution of the ultrasound-responsive microbubbles.

13. The method of claim 1, wherein:

the inert gas is a perfluorocarbon-based gas.

14. The method of claim 13, wherein:

as the perfluorocarbon-based gas, at least one of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, and perfluoroperhydrobenzyltetralin is used.

15. The method of claim 1, wherein:

in the step (b) of the present invention,

after dissolving a second mixture powder including a second lipid in an organic solvent, removing the organic solvent to obtain a lipid film, dissolving the lipid film in a second solvent to produce a solution of a second shell material, mixing the solution of the second shell material with the ultrasound-responsive microbubbles and the drug, and then irradiating ultrasound to produce the liposomes.

16. The method of claim 15, wherein:

the second lipid comprises DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), HSPC (phosphatidylcholine), DDPC (1, 2-didecanoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-di (cis-13-di-erucyl) -sn-glycero-3-phosphorylcholine), DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine), DMPC (1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine), DLPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-dilauroyl-sn-glycero-3-phosphorylcholine), DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphorylcholine), egg PC (phosphorylcholine), DPPA (diphenylphosphoryl azide), DMPA-Na (1, 2-dimyristoyl-sn-glycero-3-phosphate), DPPA-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphate), DOPA-Na (1, 2-dioleoyl-sn-glycero-3-phosphate), DSPE (distearoylphosphatidylethanolamine), DMPE (dimyristoyl phosphatidylethanolamine), DOPE (dioleoyl phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE-glutaryl- (Na)2(1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine), eg PE (phosphatidylethanolamine), DSPG (distearoyl phosphatidylglycerol), DMPG-Na (1, 2-dimyristoyl-sn-glycerol-3-phosphoglycerol), DPPG-Na (1, 2-dipalmitoyl-sn-glycerol-3-phosphoglycerol), DOPG-Na (1, 2-dioleoyl-sn-glycerol-3-phosphoglycerol), DOPS (dimyristoyl phosphatidylserine), DMPS-Na (1, 2-dimyristoyl-sn-glycero-3-phosphoserine), DPPS-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphoserine), DOPS-Na (1, 2-dioleoyl-sn-glycero-3-phosphoserine), DSPS (distearoylphosphatidylserine), DSPE-mPEG (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-maleimide PEG-2000-Na, surfactant (b): at least one of Tween 80, Span 80, dipotassium glycyrrhizinate.

17. The method of claim 16, wherein:

the second lipid comprises the DPPC and the DPPA.

18. The method of claim 16, wherein:

the second lipid comprises at least one of the DPPA, the DMPA-Na, the DPPA-Na, the DSPG, and the DSPS to charge the liposomes.

19. The method of claim 15, wherein:

the second mixture powder further includes at least one of albumin, a polymer, cholesterol, and PEG.

20. The method of claim 15, wherein:

the second solvent is PBS.

21. The method of claim 15, wherein:

the organic solvent is a mixed solvent of chloroform and methanol.

22. The method of claim 1, wherein:

in the step (b) of the present invention,

by filtering the liposomes with a filter and the extruder, the size distribution of the liposomes is uniformly formed.

23. The method of claim 15, wherein:

after irradiating ultrasonic waves to the solution of the second shell material, the microbubbles and the drug are mixed to produce the liposomes.

24. The method of claim 1, wherein:

the liposome further comprises a gene within its interior.

25. A liposome produced according to the method of any one of claims 1 to 24.

Technical Field

The present invention relates to a method for preparing a liposome including an ultrasound-responsive microbubble for delivering a drug and a drug, and a liposome using the same.

More particularly, the present invention relates to a method for preparing a liposome including ultrasound-responsive microbubbles for delivering a drug, wherein the method comprises: (a) uniformly forming a size distribution of the ultrasound-responsive microbubbles by an extruder after generating the ultrasound-responsive microbubbles including the inert gas inside and formed with the first shell outside; and (b) after liposomes comprising the ultrasound-responsive microbubbles and the drug having a uniform internal size distribution and formed with the second shell on the outside are produced, uniformly forming the size distribution of the liposomes by an extruder.

Background

A Drug Delivery System (DDS) may refer to a dosage form (dosageformulation) that can effectively deliver a required amount of Drug for treating a disease by minimizing side effects of existing drugs and optimizing the efficacy and effect of the drugs.

These drug delivery systems include transdermal, oral, or transvascular methods, and the like, depending on the drug delivery route. In addition, a drug delivery system in which a microcapsule is introduced into a blood vessel to treat an affected part is attracting attention as a next-generation treatment technology.

Further, in the technology of the drug delivery system, it can be said that the elemental technology is a technology for accurately targeting the drug to the target affected part and a technology for controlling the release of the drug in the affected part. Therefore, a targeted drug delivery system using ultrasonic waves and ultrasound-responsive microbubbles has recently attracted more attention as a technique that can solve these problems.

In particular, according to the research results, microbubbles used as ultrasound contrast agents have a cavitation effect (cavitation) caused by ultrasonic energy and the effect enhances the delivery effect of drugs to the skin or the inside of cells, so those skilled in the art have attempted to deliver drugs to the human body by binding (ligand binding) a desired drug or receptor (receptor) ligand to the membrane of the microbubble.

However, since this method binds the drug to the membrane surface, there is a limitation in that loss of the drug may occur during the movement of the microbubbles to the target site, thereby failing to fully exert the function of the drug delivery body. In addition, there is a limitation that a large amount of drug cannot be loaded.

To improve this, a technique for preparing liposomes to be simultaneously loaded with microbubbles and a drug to increase ultrasonic energy and responsiveness has recently appeared.

However, the method of simultaneously loading microbubbles including inert gas and drug into the space between liposome shells (shells) has a disadvantage that it is difficult to form a multi-layered structure and the drug is not effectively loaded.

That is, depending on the size of microbubbles trapped inside liposomes and the characteristics of the drug, the amount of drug loaded may vary, and even if severe, it may be impossible to load the drug or microbubbles into the liposomes.

Disclosure of Invention

Technical problem

The present invention aims to solve all the above problems.

In addition, another object of the present invention is to encapsulate a drug inside liposome to protect the drug from the external environment.

In addition, another object of the present invention is to prevent the generation of drug action in normal tissues and to exhibit high responsiveness to ultrasonic energy, so that the drug can be delivered by reacting only in a target region where ultrasonic energy is irradiated.

In addition, another object of the present invention is to quantify the drug loading inside liposomes by forming microbubbles and liposomes of a certain size.

Furthermore, it is another object of the present invention that a drug can be loaded in an amount of more than a certain amount to exhibit a significant drug action.

Technical scheme

A representative configuration of the present invention for achieving the above object is as follows.

The present invention provides a method for preparing a liposome comprising ultrasound-responsive microbubbles for delivering a drug, wherein the method comprises: (a) uniformly forming a size distribution of the ultrasound-responsive microbubbles by an extruder after generating the ultrasound-responsive microbubbles including the inert gas inside and formed with the first shell outside; and (b) after liposomes comprising the ultrasound-responsive microbubbles and the drug having a uniform internal size distribution and formed with the second shell on the outside are produced, uniformly forming the size distribution of the liposomes by an extruder.

In addition, according to an embodiment of the present invention, in a method of preparing a liposome including an ultrasound-responsive microbubble for delivering a drug, there is further provided a liposome including an ultrasound-responsive microbubble for delivering a drug, including: an ultrasound-responsive microbubble in which, after the ultrasound-responsive microbubble in which an inert gas is included inside and a first shell is formed outside is generated, a size distribution of the ultrasound-responsive microbubble is uniformly formed by an extruder; and liposomes, wherein, after the liposomes including the ultrasound-responsive microbubbles and the drug having a uniform inner size distribution and formed with the second shell on the outside are produced, the size distribution of the liposomes is uniformly formed by an extruder.

Advantageous effects

The effects according to the invention are as follows:

the present invention protects the drug from the external environment by encapsulating the drug inside liposomes.

In addition, the present invention prevents the generation of drug action in normal tissues and exhibits high responsiveness to ultrasonic energy, so that the drug can be delivered by reacting only in the target region where ultrasonic energy is irradiated.

In addition, the present invention can quantify the drug loading inside the liposomes by forming microbubbles and liposomes of a certain size.

In addition, the present invention can be loaded with a certain amount or more of a drug to exhibit a significant drug action.

Drawings

Fig. 1 is a schematic diagram of a liposome including ultrasound-responsive microbubbles for delivering a drug and a drug, according to an embodiment of the invention.

Fig. 2 is a state diagram for adjusting the size of microbubbles according to an embodiment of the invention.

FIG. 3 is a schematic diagram of a confocal microscope image of microbubbles according to an embodiment of the invention.

Fig. 4a to 4c are graphs showing the results of analyzing the sizes of microbubbles by intensity (intensity), volume (volume) and number (number) distribution according to an embodiment of the present invention.

Fig. 5 is a state diagram for adjusting the size of liposomes according to an embodiment of the present invention.

Fig. 6 is a confocal microscopy analysis image of liposomes according to one embodiment of the invention.

Fig. 7 is a result of comparing confocal microscopy analysis images of liposomes according to an embodiment of the present invention with liposomes prepared by a prior art method.

Fig. 8 is a state diagram of a gold nanoparticle trapping experiment using liposomes according to an embodiment of the present invention.

Detailed Description

For the following detailed description of the invention, reference is made to the accompanying drawings that show, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It is to be understood that the various embodiments of the invention are different, but not necessarily mutually exclusive. For example, particular shapes, structures and characteristics described in one embodiment may be implemented in other embodiments without departing from the spirit and scope of the invention. In addition, it is to be understood that the location or arrangement of individual components within each disclosed embodiment may be modified without departing from the spirit and scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled, if appropriately interpreted. In all of the various aspects, like numerals refer to the same or similar functionality throughout the several views.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that those skilled in the art can easily implement the present invention.

Fig. 1 is a schematic diagram of a liposome including ultrasound-responsive microbubbles 11 for delivering a drug, according to an embodiment of the present invention.

Referring to fig. 1, in the liposome, microbubbles 11 are formed inside and a second shell 22 is formed outside. And the drug may be loaded in the region 21 between the microbubbles 11 and the second shell 22. In addition, the first shell 12 may be formed on the outer surface of the microbubbles 11.

Liposomes of this structure are made by the following process: first, microbubbles 11 having a uniform size distribution according to an embodiment of the present invention are generated, then liposomes including the microbubbles and the drug inside and having the second shell formed on the outside are generated, and the size distribution of the liposomes is uniformly formed by an extruder.

The process of generating the ultrasound-responsive microbubbles 11 will be described in detail below.

First, a solution of the first shell material for preparing the microbubbles 11 is prepared.

To this end, a first mixture powder including a first lipid may be dissolved in a first solvent to produce a solution of a first shell material. Wherein the first mixture powder including the first lipid may further include albumin, a polymer, PEG, a surfactant, a protein, a biodegradable polymer, etc., and cholesterol (cholestrol) may be added to increase durability of the ultrasound-responsive microbubbles.

In addition, the first lipid may comprise DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), HSPC (phosphatidylcholine), DDPC (1, 2-didecanoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-bis (cis-13-dicaprylyl) -sn-glycero-3-phosphorylcholine), DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine), DMPC (1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine), DLPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), DEPC (1, 2-dilauroyl-sn-glycero-3-phosphorylcholine), DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphorylcholine), egg PC (phosphorylcholine), DPPA (diphenylphosphoryl azide), DMPA-Na (1, 2-dimyristoyl-sn-glycero-3-phosphate), DPPA-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphate), DOPA-Na (1, 2-dioleoyl-sn-glycero-3-phosphate), DSPE (distearoylphosphatidylethanolamine), DMPE (dimyristoyl phosphatidylethanolamine), DOPE (dioleoyl phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE-glutaryl- (Na)2(1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine), eg PE (phosphatidylethanolamine), DSPG (distearoyl phosphatidylglycerol), DMPG-Na (1, 2-dimyristoyl-sn-glycerol-3-phosphoglycerol), DPPG-Na (1, 2-dipalmitoyl-sn-glycerol-3-phosphoglycerol), DOPG-Na (1, 2-dioleoyl-sn-glycerol-3-phosphoglycerol), DOPS (dimyristoyl phosphatidylserine), DMPS-Na (1, 2-dimyristoyl-sn-glycero-3-phosphoserine), DPPS-Na (1, 2-dipalmitoyl-sn-glycero-3-phosphoserine), DOPS-Na (1, 2-dioleoyl-sn-glycero-3-phosphoserine), DSPS (distearoylphosphatidylserine), DSPE-mPEG (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-maleimide PEG-2000-Na, surfactant (b): at least one of Tween 80, Span 80, dipotassium glycyrrhizinate.

In addition, albumin may include serum albumin (serum albumin), ovalbumin (ovalbumin), and the like.

In addition, the polymer may include PBLA (poly (. beta. -benzyl-L-aspartic acid)), PDLA (poly-DL-lactic acid), and the like.

In addition, the surfactant may include sodium fatty acid, monoalkyl sulfate, alkylpolyoxyethyl sulfate, alkylbenzene sulfonate, monoalkyl phosphate, dialkyldimethyl ammonium salt, alkylbenzylmethyl ammonium salt, alkylsulfobetaine, alkylcarboxyl betaine, polyoxyethylene alkyl ether, fatty acid sorbitol ester, fatty acid diethanolamine, alkylmonoglyceryl ether, benzalkonium chloride (benzalkonium chloride), benzethonium chloride (benzethonium chloride), and the like.

In addition, the protein may include albumin, globulin, collagen, and the like.

In addition, the biodegradable polymer may include PHB-based plastic, polysaccharide-based plastic, Polycaprolactone (PCL), polylactic acid (PLA), poly (propyleneglycolic acid) (PG), polyhydroxybutyrate-co-valerate (PHBV), polyvinyl alcohol (PVA), polybutylene succinate (PBS), chitin-based plastic, oil-based plastic, and the like.

Additionally, the first solvent may include a salt solution and/or at least one of tri-distilled water, glycerol, and propylene glycol.

As an example, after mixing materials of lipid (lipid), albumin (albumin), polymer (polymer), cholesterol (cholestrol), peg (polyethylene glycol), and the like constituting the powdered shell with a solvent including a salt solution and/or at least one of triple distilled water (40% to 60%), glycerin (2% to 10%), propylene glycol (40% to 60%), a solution of the first shell material may be produced by dissolving at a temperature between 60 ℃ and 100 ℃ for 1 hour to 6 hours.

As an example, micelles of ultrasound (micella) specifically used for drug delivery use DPPC (dipalmitoyl-phosphatidyl-choline) and DPPA (diphenyl-phosphoryl-azide) as the main components of the shell material to trap inert gases and increase the stability of bubbles, and physiological saline, glycerol, and propylene glycol may be added together.

Also, when DPPC and DPPA as main components constitute the shell, cholesterol (cholestrol) may be added to increase the durability of the micelle.

Next, the solution of the first housing material and the inert gas may be mixed to increase the responsiveness to the ultrasonic energy.

At this time, the inert gas may be a perfluorocarbon-based gas, and as the perfluorocarbon-based inert gas, perfluoromethane (perfluoromethane), perfluoroethane (perfluorethane), perfluoropropane (perfluoropropane), perfluorobutane (perfluorobutane), perfluoro-n-pentane (perfluor-n-pentane), perfluorohexane (perfluor-n-hexane), perfluoromethylcyclopentane (perfluoromethylcyclopentane), perfluoro-1,3-dimethylcyclohexane (perfluor-1, 3-dimethylcyclohexane), perfluorodecalin (perfluorodecin), perfluoromethyldecalin (perfluoromethyldecalin), perfluoroperhydrobenzyltetralin (perfluorperhydrobenzyltetralin), or the like may be used.

As an example, a solution of the first casing material and an inert gas are mixed in a ratio of 1: 1 to 20: 1 (v/v) was dispensed into a vial and sealed, and then the housing material and inert gas were mechanically mixed by a vial mixer.

At this time, the mechanical mixing speed may be adjusted to 1000rpm to 5000rpm to appropriately control the size and particle size distribution of the ultrasound-responsive microbubbles 11.

As such, the perfluorocarbon-based inert gas is finely pulverized into nano-to micro-sized oil/water emulsion by mechanical mixing, and the inert gas can be naturally combined with the hydrophobic tail of the amphiphilic phospholipid by self-assembly (self-assembly) to maintain a stable state, thereby forming microbubbles 11 having the inert gas as a core inside as shown in fig. 1. Specifically, the fatty acid chain corresponding to the tail portion in the phospholipid is hydrophobic, and the phosphate and base portion as the head portion has hydrophilic amphiphilicity. These amphiphilic (amphothic) phospholipids, which have both hydrophilic and hydrophobic properties, play an important role in building the shell. In addition, the microbubbles 11 may be ultrasound-responsive bubbles.

Next, as shown in fig. 2, the ultrasound-responsive microbubbles 11 prepared in various sizes can be filtered by using a filter having a certain pore size, for example, any one pore size of 30nm to 1um and an extruder, so that the size distribution of the ultrasound-responsive microbubbles is uniform. At this time, the filter may be a membrane filter, and the membrane filter may be formed of polycarboxylates.

In addition, the temperature for filtering the ultrasound-responsive microbubbles 11 can be variously adjusted from room temperature to the phase transition temperature of each material, and the number of times of filtering can be performed at least 5 times to more than that.

Subsequently, the mixture including the ultrasound-responsive microbubbles 11 uniformly formed in size distribution by filtration is centrifuged with a centrifuge to settle well-formed micelles, and then the supernatant present in the upper layer is removed and washed with deionized water (deionized water), so that the ultrasound-responsive microbubbles adjusted to a desired size can be obtained.