KR102396305B1 - Method for preparing liposome synthesis containing ultrasound responsive microbubble for drug delivery and liposome using the same - Google Patents

Method for preparing liposome synthesis containing ultrasound responsive microbubble for drug delivery and liposome using the same Download PDF

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KR102396305B1
KR102396305B1 KR1020210102819A KR20210102819A KR102396305B1 KR 102396305 B1 KR102396305 B1 KR 102396305B1 KR 1020210102819 A KR1020210102819 A KR 1020210102819A KR 20210102819 A KR20210102819 A KR 20210102819A KR 102396305 B1 KR102396305 B1 KR 102396305B1
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liposome
microbubbles
drug
size distribution
glycerol
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KR20210100061A (en
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김철우
박동희
원종호
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(주)바이오인프라생명과학
주식회사 퍼시픽시스템
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Abstract

약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및 (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;를 포함하는 방법 및 이를 이용한 리포좀을 제공한다.In the method for producing a liposome containing ultrasonically responsive microbubbles for drug delivery, (a) after generating ultrasonically responsive microbubbles containing an inert gas inside and having a first shell formed on the outer surface, the ultrasonic wave through the extruder uniformly forming a size distribution of reactive microbubbles; and (b) forming a liposome containing the ultrasonically responsive microbubbles and a drug having a uniform size distribution therein and having a second shell formed on the outer surface, followed by uniformly forming the size distribution of the liposome through an extruder; It provides a method comprising a, and liposomes using the same.

Description

약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀 제조 방법 및 이를 이용한 리포좀{METHOD FOR PREPARING LIPOSOME SYNTHESIS CONTAINING ULTRASOUND RESPONSIVE MICROBUBBLE FOR DRUG DELIVERY AND LIPOSOME USING THE SAME}Liposome manufacturing method including ultrasonic responsive microbubbles for drug delivery, and liposome using the same

본 발명은 약물전달을 위한 초음파 반응형 미소기포와 약물을 포함하는 리포좀의 제조 방법 및 이를 이용한 리포좀에 관한 것이다.The present invention relates to a method for preparing a liposome containing ultrasonically responsive microbubbles and a drug for drug delivery, and to a liposome using the same.

보다 상세하게는, 약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및 (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;를 포함하는 방법 및 이를 이용한 리포좀에 관한 것이다.More specifically, in the method for producing a liposome containing ultrasonically responsive microbubbles for drug delivery, (a) after generating ultrasonically responsive microbubbles containing an inert gas inside and having a first shell formed on the outer surface uniformly forming a size distribution of the ultrasonically responsive microbubbles through an extruder; and (b) forming a liposome containing the ultrasonically responsive microbubbles and a drug having a uniform size distribution therein and having a second shell formed on the outer surface, followed by uniformly forming the size distribution of the liposome through an extruder; It relates to a method comprising a and liposomes using the same.

약물전달시스템(DDS: Drug Delivery System)은 기존의 약물을 부작용을 최소화하고 약물이 가지고 있는 효능 및 효과를 최적화하여 질병치료를 위하여 필요한 양을 약물을 효율적으로 전달하기 위한 제형(dosage formulation)이라 할 수 있다.A drug delivery system (DDS) is a dosage formulation for efficiently delivering the drug in the amount required for disease treatment by minimizing the side effects of the existing drug and optimizing the efficacy and effect of the drug. can

이러한 약물전달시스템은 약물전달경로에 따라서 경피, 경구 또는 혈관을 통한 방법 등이 있다. 또한 마이크로 크기의 캡슐을 혈관에 도입하여 환부를 치료하는 약물전달시스템이 앞으로 꿈의 치료기술로써 각광을 받고 있다.Such drug delivery systems include transdermal, oral, or blood vessel methods depending on the drug delivery route. In addition, a drug delivery system that treats the affected area by introducing micro-sized capsules into the blood vessels is in the spotlight as a dream treatment technology in the future.

그리고 약물전달시스템의 기술 중에서 요소기술은 약물을 목적하는 환부에 정확히 타겟팅하는 기술과 환부에서의 약물방출을 제어하는 기술이라고 할 수 있다. 따라서 초음파와 초음파 반응형 미소기포에 의한 표적약물전달 시스템은 이러한 문제점들을 해결할 수 있는 기술로써, 최근에 더욱 관심을 모으고 있다.And among the technologies of the drug delivery system, the elemental technology can be said to be a technology to precisely target a drug to a target affected area and a technology to control the drug release from the affected area. Therefore, a targeted drug delivery system using ultrasound and ultrasound-responsive microbubbles is a technology that can solve these problems, and has recently attracted more attention.

특히, 초음파 조영제로 사용되는 미소기포는 초음파 에너지에 의해 공동화 현상(cavitation)이 발생하고 이 현상은 피부나 세포 내부로의 약물전달 효과를 증가시킨다는 연구 결과에 따라 미소기포의 막에 원하는 약물이나 수용체(receptor)를 리간드 결합(ligand binding)하여 약물을 인체에 전달하고자 하였다.In particular, according to the research results that the microbubbles used as ultrasound contrast agents cause cavitation by ultrasonic energy, and this phenomenon increases the effect of drug delivery into the skin or cells, the desired drug or receptor on the membrane of the microbubbles (Receptor) was intended to be delivered to the human body by ligand binding (ligand binding).

그러나, 이러한 방법은 막 표면에 약물을 결합시키므로, 미소기포가 타겟 위치까지 이동하는 중에 약물의 유실이 발생할 수 있어 약물 전달체의 역할을 완벽하게 수행할 수 없다는 한계가 있다. 또한, 많은 양의 약물을 탑재할 수 없다는 점에서 한계가 있다.However, since this method binds the drug to the membrane surface, drug loss may occur while the microbubbles move to the target location, and thus there is a limitation in that it cannot perfectly perform the role of the drug carrier. In addition, there is a limitation in that a large amount of drug cannot be loaded.

이를 개선하기 위하여, 최근에는 초음파 에너지와 반응성을 높이기 위한 미소기포와 약물을 동시에 탑재한 리포좀 제조 기술이 대두되고 있다.In order to improve this, recently, a technology for preparing liposomes in which microbubbles and drugs are simultaneously loaded to increase ultrasonic energy and reactivity has emerged.

하지만, 불활성 가스를 함유한 미소기포와 약물을 리포좀 막(shell) 사이 공간에 동시에 탑재하는 방식의 제조법은 다층 구조를 형성하기 어려우며 또한 효과적으로 약물을 탑재하지 못한다는 단점이 있다.However, the method of simultaneously loading microbubbles and drugs containing inert gas in the space between the liposome shells has disadvantages in that it is difficult to form a multi-layered structure, and the drugs cannot be loaded effectively.

즉, 리포좀의 내부에 포집되는 미소기포의 크기 및 약물의 특성에 따라 탑재되는 약물의 양이 서로 다르게 되며, 심한 경우 리포좀에 약물이 탑재되지 못하거나 미소기포가 탑재되지 못하는 경우가 발생한다.That is, the amount of drug loaded varies depending on the size of the microbubbles collected inside the liposome and the properties of the drug, and in severe cases, the drug cannot be loaded on the liposome or the microbubbles cannot be loaded.

본 발명은 상술한 문제점들을 모두 해결하는 것을 그 목적으로 한다.An object of the present invention is to solve all of the above problems.

또한, 본 발명은 약물을 리포좀 내부로 캡슐화하여 외부 환경으로부터 약물을 보호할 수 있도록 하는 것을 다른 목적으로 한다.Another object of the present invention is to protect the drug from the external environment by encapsulating the drug into the liposome.

또한, 본 발명은 정상 조직에서의 약효 발생을 차단하며 초음파 에너지에 높은 반응성을 나타내어 초음파 에너지가 조사되고 있는 표적 영역에서만 상호 반응하여 약물을 전달할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to block the occurrence of drug effects in normal tissues and to exhibit high reactivity to ultrasonic energy so that the drug can be delivered by mutual reaction only in the target region to which ultrasonic energy is irradiated.

또한, 본 발명은 미소기포 및 리포좀의 크기를 일정하게 형성함으로써 리포좀 내부에 탑재되는 약물의 양을 정량화할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to quantify the amount of a drug loaded inside the liposome by forming the microbubbles and liposomes in a constant size.

또한, 본 발명은 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물을 탑재할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, it is another object of the present invention to be able to load a certain amount or more of a drug to exhibit a significant drug effect.

상기 목적을 달성하기 위한 본 발명의 대표적인 구성은 다음과 같다.A representative configuration of the present invention for achieving the above object is as follows.

약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및 (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;를 포함하는 방법이 제공된다.In the method for producing a liposome containing ultrasonically responsive microbubbles for drug delivery, (a) after generating ultrasonically responsive microbubbles containing an inert gas inside and having a first shell formed on the outer surface, the ultrasonic wave through the extruder uniformly forming a size distribution of reactive microbubbles; and (b) forming a liposome containing the ultrasonically responsive microbubbles and a drug having a uniform size distribution therein and having a second shell formed on the outer surface, followed by uniformly forming the size distribution of the liposome through an extruder; A method comprising:

또한, 본 발명의 일 실시예에 따르면, 약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포가 생성된 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포; 및 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀이 생성된 이후 압출기를 통해 상기 리포좀의 크기 분포가 균일하게 형성된 상기 리포좀;을 포함하는 약물 전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀을 제공한다.In addition, according to an embodiment of the present invention, in the method for producing a liposome containing ultrasonically responsive microbubbles for drug delivery, the ultrasonically responsive microbubbles containing an inert gas inside and having a first shell formed on the outer surface are the ultrasonically responsive microbubbles in which the size distribution of the ultrasonically responsive microbubbles is uniformly formed through an extruder after being generated; and the liposome in which the size distribution of the liposome is uniformly formed through an extruder after the liposome including the ultrasonically responsive microbubbles and the drug having a uniform size distribution therein and having a second shell formed on the outer surface is generated. Provided is a liposome comprising ultrasonically responsive microbubbles for drug delivery.

본 발명에 의하면, 다음과 같은 효과가 있다.According to the present invention, the following effects are obtained.

본 발명은 약물을 리포좀 내부로 캡슐화 하여 외부 환경으로부터 약물을 보호할 수 있다.The present invention can protect the drug from the external environment by encapsulating the drug inside the liposome.

또한, 본 발명은 정상 조직에서의 약효 발생을 차단하며 초음파 에너지에 높은 반응성을 나타내어 초음파 에너지가 조사되고 있는 표적 영역에서만 상호 반응하여 약물을 전달할 수 있다.In addition, the present invention blocks the occurrence of drug effects in normal tissues and exhibits high reactivity to ultrasonic energy, so that the drug can be delivered by mutual reaction only in the target region irradiated with ultrasonic energy.

또한, 본 발명은 미소기포 및 리포좀의 크기를 일정하게 형성함으로써 리포좀 내부에 탑재되는 약물의 양을 정량화할 수 있다.In addition, the present invention can quantify the amount of the drug loaded inside the liposome by forming the size of the microbubbles and liposomes constant.

또한, 본 발명은 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물을 탑재할 수 있다.In addition, the present invention can load a certain amount or more of a drug to exhibit a significant drug effect.

도 1은 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포와 약물을 함유한 리포좀을 개략적으로 도시한 것이고,
도 2는 본 발명의 일 실시예에 따른 미소기포의 사이즈를 조절하는 상태를 개략적으로 도시한 것이고,
도 3은 본 발명의 일 실시예에 따른 미소기포의 공초점 현미경 영상을 개략적으로 도시한 것이고,
도 4는 본 발명의 일 실시예에 따른 미소기포의 입도를 강도(intensity), 용적(volume), 개수(number) 분포로 분석한 결과를 개략적으로 도시한 것이고,
도 5는 본 발명의 일 실시예에 따른 리포좀의 사이즈를 조절하는 상태를 개략적으로 도시한 것이고,
도 6은 본 발명의 일 실시예에 따른 리포좀에 대한 공초점 현미경 분석 이미지를 도시한 것이고,
도 7은 본 발명의 일 실시예에 따른 리포좀과 종래 방법에 의해 제작된 리포좀에 대한 공초점 현미경 분석 이미지를 각각 비교하여 도시한 것이고,
도 8은 본 발명의 일 실시예에 따른 리포좀을 이용하여 골드 나노 파티클 포집 실험을 수행한 상태를 개략적으로 도시한 것이다.1 schematically shows a liposome containing ultrasound-responsive microbubbles and a drug for drug delivery according to an embodiment of the present invention;
Figure 2 schematically shows a state of adjusting the size of microbubbles according to an embodiment of the present invention,
Figure 3 schematically shows a confocal microscope image of microbubbles according to an embodiment of the present invention,
Figure 4 schematically shows the results of analyzing the particle size of microbubbles in terms of intensity, volume, and number distribution according to an embodiment of the present invention;
Figure 5 schematically shows the state of adjusting the size of the liposome according to an embodiment of the present invention,
Figure 6 shows a confocal microscopy image of the liposome according to an embodiment of the present invention,
7 is a view showing a comparison of each of the confocal microscopic analysis images of the liposome according to an embodiment of the present invention and the liposome prepared by the conventional method,
FIG. 8 schematically shows a state in which a gold nanoparticle collection experiment was performed using a liposome according to an embodiment of the present invention.

후술하는 본 발명에 대한 상세한 설명은, 본 발명이 실시될 수 있는 특정 실시예를 예시로서 도시하는 첨부 도면을 참조한다. 이들 실시예는 당업자가 본 발명을 실시할 수 있기에 충분하도록 상세히 설명된다. 본 발명의 다양한 실시예는 서로 다르지만 상호 배타적일 필요는 없음이 이해되어야 한다. 예를 들어, 여기에 기재되어 있는 특정 형상, 구조 및 특성은 일 실시예에 관련하여 본 발명의 정신 및 범위를 벗어나지 않으면서 다른 실시예로 구현될 수 있다. 또한, 각각의 개시된 실시예 내의 개별 구성요소의 위치 또는 배치는 본 발명의 정신 및 범위를 벗어나지 않으면서 변경될 수 있음이 이해되어야 한다. 따라서, 후술하는 상세한 설명은 한정적인 의미로서 취하려는 것이 아니며, 본 발명의 범위는, 적절하게 설명된다면, 그 청구항들이 주장하는 것과 균등한 모든 범위와 더불어 첨부된 청구항에 의해서만 한정된다. 도면에서 유사한 참조부호는 여러 측면에 걸쳐서 동일하거나 유사한 기능을 지칭한다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0012] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0010] Reference is made to the accompanying drawings, which show by way of illustration specific embodiments in which the present invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present invention. It should be understood that the various embodiments of the present invention are different but need not be mutually exclusive. For example, certain shapes, structures, and characteristics described herein with respect to one embodiment may be implemented in other embodiments without departing from the spirit and scope of the invention. In addition, it should be understood that the location or arrangement of individual components within each disclosed embodiment may be changed without departing from the spirit and scope of the present invention. Accordingly, the detailed description set forth below is not intended to be taken in a limiting sense, and the scope of the present invention, if properly described, is limited only by the appended claims, along with all scope equivalents as those claimed. Like reference numerals in the drawings refer to the same or similar functions throughout the various aspects.

이하, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 본 발명을 용이하게 실시할 수 있도록 하기 위하여, 본 발명의 바람직한 실시예들에 관하여 첨부된 도면을 참조하여 상세히 설명하기로 한다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to enable those of ordinary skill in the art to easily practice the present invention.

도 1은 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포(11)를 포함하는 리포좀을 개략적으로 도시한 것이다.1 schematically shows a liposome including ultrasonically responsive microbubbles 11 for drug delivery according to an embodiment of the present invention.

도 1을 참조하면, 리포좀은 내부에 미소기포(11)가 형성되며 외면에 제2쉘(22)이 형성될 수 있다. 그리고, 미소기포(11)와 제2쉘(22)의 사이 영역(21)에는 약물이 탑재될 수 있다. 또한, 미소기포(11)의 외면에는 제1쉘(12)이 형성될 수 있다.Referring to FIG. 1 , the liposome may have microbubbles 11 formed therein and a second shell 22 may be formed on the outer surface thereof. In addition, 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 .

이와 같은 구조의 리포좀은 본 발명의 일 실시예에 의해 크기 분포가 균일하게 형성된 미소기포(11)를 먼저 생성한 다음, 내부에 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성하고, 압출기를 통해 리포좀의 크기 분포를 균일하게 형성하는 과정을 거쳐 만들어지게 된다.The liposome having such a structure first generates microbubbles 11 having a uniform size distribution according to an embodiment of the present invention, and then contains microbubbles and a drug inside, and creates a liposome having a second shell formed on the outer surface. And it is made through the process of uniformly forming the size distribution of the liposomes through the extruder.

아래에서는 초음파 반응형 미소기포(11)를 생성하는 과정에 대해서 구체적으로 설명한다.Hereinafter, the process of generating the ultrasonically responsive microbubbles 11 will be described in detail.

먼저, 미소기포(11)를 제조하기 위한 제1쉘 물질의 솔루션을 준비한다.First, a solution of the first shell material for manufacturing the microbubbles 11 is prepared.

이를 위해 제1 리피드를 포함하는 제1 혼합물 파우더를 제1 용매에 용해하여 제1쉘 물질의 솔루션을 생성할 수 있다. 여기서, 제1 리피드가 포함된 제1 혼합물 파우더에는 알부민, 폴리머, PEG, 계면활성제, 단백질, 생분해성 고분자 등이 더 포함될 수 있으며, 초음파 반응형 미소기포의 내구성을 높여주기 위해 콜레스테롤(cholesterol)이 첨가될 수 있다.To this end, a solution of the first shell material may be generated by dissolving the first mixture powder including the first lipid in the first solvent. Here, the first mixture powder containing the first lipid may further include albumin, polymer, PEG, surfactant, protein, biodegradable polymer, etc., and cholesterol is added to increase the durability of ultrasonically responsive microbubbles. may be added.

또한, 제1 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg 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(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함할 수 있다.In addition, the first lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3) -phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA ( Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2- Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Diolephosphatidylethanolamine) -glycerol -3-phosphoethanolamine), egg 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 (ioleoyl phosphatidylserine), DMPS (Dimyristoyl phosphatidylserine), DMPS-Na (1,2-Dimyristoyl-sn-glycerol) -3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE -mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3- phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, and at least one of dipotassium glycyrrhizinate may be included.

또한, 알부민은 세럼 알부민 (serum albumin), 오발부민 (ovalbumin) 등을 포함할 수 있다.In addition, the albumin may include serum albumin, ovalbumin, and the like.

또한, 폴리머는 Poly(β-benzyl-L-asparate), PBLA(poly-DL-lactic acid) 등을 포함할 수 있다.In addition, the polymer may include poly(β-benzyl-L-asparate), poly-DL-lactic acid (PBLA), and the like.

또한, 계면활성제는 지방산 나트륨, 모노알킬 황산염, 알킬폴리옥시에틸렌 황산염, 알킬벤젠술폰산염, 모노알킬인산염, 디알킬디메틸암모늄염, 알킬벤질메틸암모늄염, 알킬설포베타인, 알킬카르복시베타인, 폴리옥시에틸렌알킬에테르, 지방산 솔비탄에스테르, 지방산 디에탄올아민, 알킬모노글리세릴에테르, 벤잘코늄 염화물 (benzalkonium chloride), 벤제토늄 염화물 (benzethonium chloride) 등을 포함할 수 있다.In addition, surfactants include sodium fatty acid, monoalkyl sulfate, alkylpolyoxyethylene sulfate, alkylbenzenesulfonate, monoalkylphosphate, dialkyldimethylammonium salt, alkylbenzylmethylammonium salt, alkylsulfobetaine, alkylcarboxybetaine, polyoxyethylene It may include alkyl ether, fatty acid sorbitan ester, fatty acid diethanolamine, alkyl monoglyceryl ether, benzalkonium chloride, benzethonium chloride, and the like.

또한, 단백질은 알부민, 글로불린, 콜라겐 등을 포함할 수 있다.In addition, the protein may include albumin, globulin, collagen, and the like.

또한, 생분해성 고분자는 PHB계 플라스틱, 다당류계 플라스틱,　폴리카프로락톤(PCL),　폴리유산(PLA),　폴리필렌 글리콜산(PG), 폴리리드록시부티레이트-코-발레레이트(PHBV), 폴리비닐알콜(PVA), 폴리부틸렌 숙시네이트(PBS), Chitin계 플라스틱, Oil계 플라스틱 등을 포함할 수 있다.In addition, biodegradable polymers include PHB-based plastics, polysaccharide-based plastics, 　polycaprolactone (PCL), 　polylactic acid (PLA), 　polyfillene glycolic acid (PG), polyhydroxybutyrate-co-valerate (PHBV), polyvinyl. Alcohol (PVA), polybutylene succinate (PBS), chitin-based plastics, oil-based plastics, and the like may be included.

또한, 제1 용매에는 Saline 및/또는 3차 증류수, glycerin 및 propylene glycol 중 적어도 하나 이상이 포함될 수 있다.In addition, the first solvent may include at least one of saline and/or tertiary distilled water, glycerin, and propylene glycol.

일 예로, 파우더 상태의 쉘을 구성하는 리피드(lipid), 알부민(albumin), 폴리머(polymer), 콜레스테롤(cholesterol) 및 PEG(polyethylene glycol) 등의 물질을 Saline 및/또는 3차 증류수(40~60%), glycerin(2~10%), propylene glycol(40~60%) 중 하나 이상을 포함하는 용매와 혼합한 뒤 60°C~100°C 사이의 온도에서 1~6시간 동안 녹여줌으로써 제1쉘 물질의 솔루션을 생성할 수 있다 .For example, substances such as lipid, albumin, polymer, cholesterol, and PEG (polyethylene glycol) constituting the powder-state shell are mixed with Saline and/or tertiary distilled water (40 to 60 %), glycerin (2-10%), and propylene glycol (40-60%) mixed with a solvent containing at least one A solution of shell material can be produced.

일 예로, 약물 전달을 위한 초음파에 특화된 미셀(micelle)은 불활성 가스를 포집하고 기포의 안정성을 높이기 위해 쉘 물질로 DPPC (Dipalmitoyl -Phos Phatidyl - Choline) 및 DPPA(Diphenyl-phosphoryl-Azide)를 주요 구성물로 하고 Normal saline, Glycerol, Propylene glycol이 함께 첨가될 수 있다.For example, micelles specialized in ultrasound for drug delivery include DPPC (Dipalmitoyl-Phos Phatidyl-Choline) and DPPA (Diphenyl-phosphoryl-Azide) as a shell material to capture inert gas and increase the stability of bubbles. and Normal saline, Glycerol, and Propylene glycol may be added together.

그리고, 주요 구성물인 DPPC와 DPPA가 쉘을 구성할 때 미셀의 내구성을 높여주기 위해 콜레스테롤(cholesterol)이 첨가될 수 있다.And, when DPPC and DPPA, which are the main components, constitute the shell, cholesterol may be added to increase the durability of micelles.

다음으로, 초음파 에너지에 반응성을 높이기 위해 제1쉘 물질의 솔루션과 불활성 가스를 혼합할 수 있다.Next, in order to increase the reactivity to ultrasonic energy, a solution of the first shell material and an inert gas may be mixed.

이때, 불활성 가스는 퍼플루오로카본 계열의 가스일 수 있으며, 퍼플루오로카본 계열의 불활성 가스로서 Perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perfluoroperhydrobenzyltetralin 등을 사용할 수 있다.In this case, the inert gas may be a perfluorocarbon-based gas, and as a perfluorocarbon-based inert gas, Perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1 ,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perfluoroperhydrobenzyltetralin, etc. may be used.

일 예로, 제1쉘 물질의 솔루션과 불활성 가스의 혼합비율(v/v)을 1:1 내지 20:1의 비율로 바이얼에 분주하여 밀봉 후, 바이얼믹서를 통해 쉘 물질과 불활성 가스를 기계적 믹싱할 수 있다.For example, the mixing ratio (v/v) of the solution of the first shell material and the inert gas is dispensed into a vial at a ratio of 1:1 to 20:1 and sealed, and then the shell material and the inert gas are mixed through a vial mixer. Can be mixed mechanically.

이때, 기계적 믹싱 속도는 1000 ~ 5000 rpm으로 조절하여 초음파 반응형 미소기포(11)의 크기 및 입도 분포를 적절하게 조절할 수 있다.In this case, the mechanical mixing speed may be adjusted to 1000 ~ 5000 rpm to appropriately control the size and particle size distribution of the ultrasonically responsive microbubbles 11 .

그러면, 기계적 믹싱을 통해 퍼플루오로카본 계열의 불활성 가스가 nano 내지 micro 사이즈의 oil/water 에멀젼 형태로 잘게 부수어지며, 불활성 가스는 자기 정합(self-assembling)에 의해 자연스럽게 양친매성 인지질의 소수성 꼬리 부분과 결합되어 안정된 상태를 유지할 수 있게 되어 도 1에서와 같이 내부에 코어로서의 불활성 가스를 가지는 미소기포(11)를 형성하게 된다. 구체적으로는, 인지질 중 꼬리 부분에 해당하는 지방산 사슬은 소수성이고, 헤더 부분인 인산과 염기 부분은 친수성인 양친매성 특성을 가진다. 이런 친수성 특성과 소수성 특성을 동시에 지니는 양친매성(amphipathic) 인지질은 쉘을 구성하는데 있어 중요한 역할을 한다. 그리고, 미소기포(11)는 초음파 반응성 기포일 수 있다.Then, through mechanical mixing, the perfluorocarbon-based inert gas is crushed into nano to micro-sized oil/water emulsion, and the inert gas is naturally self-assembling by self-assembling to form the hydrophobic tail of the phospholipid. It is possible to maintain a stable state by being combined with the microbubbles 11 having an inert gas as a core therein as shown in FIG. 1 . Specifically, the fatty acid chain corresponding to the tail portion of the phospholipid is hydrophobic, and the phosphoric acid and base portion, which are the header portions, are hydrophilic and have amphiphilic properties. Amphipathic phospholipids having both hydrophilic and hydrophobic properties play an important role in constructing the shell. In addition, the microbubbles 11 may be ultrasonically responsive bubbles.

다음으로, 도 2에서와 같이, 다양한 크기로 제작된 초음파 반응형 미소기포(11)를 일정한 공극 사이즈, 일 예로 30nm 내지 1um 중 어느 하나의 공극 사이즈를 갖는 필터와 압출기를 사용하여 필터링함으로써 초음파 반응형 미소기포(11)의 크기 분포를 균일하게 할 수 있다. 이때, 필터는 멤브레인 필터일 수 있으며, 멤브레인 필터는 폴리 카보나이트로 형성될 수 있다.Next, as in FIG. 2, ultrasonic reaction by filtering the ultrasonically responsive microbubbles 11 manufactured in various sizes using a filter and an extruder having a certain pore size, for example, a pore size of any one of 30 nm to 1 μm. The size distribution of the mold microbubbles 11 can be made uniform. In this case, the filter may be a membrane filter, and the membrane filter may be formed of polycarbonite.

또한, 초음파 반응형 미소기포(11)의 필터링을 위한 온도는 상온에서 각 물질의 상전이 온도 범위까지 다양하게 조절할 수 있으며, 필터링의 횟수는 최소 5회에서 그 이상으로 수행할 수 있다.In addition, the temperature for filtering the ultrasonically responsive microbubbles 11 can be variously adjusted from room temperature to the phase transition temperature range of each material, and the number of filtering can be performed at least 5 times or more.

이후, 필터링에 의해 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)를 포함하는 혼합물을 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)를 이용하여 워싱함으로써 원하는 크기로 조정된 초음파 반응형 미소기포(11)를 획득할 수 있다.Thereafter, the mixture containing the ultrasonically responsive microbubbles 11 having a uniform size distribution by filtering is centrifuged through a centrifuge to sink the well-formed micelles, and then the supernatant present in the upper layer is removed and deionized (DW). By washing using water), it is possible to obtain the ultrasonically responsive microbubbles 11 adjusted to a desired size.

실시예1. 초음파 반응형 미소기포 제작Example 1. Ultrasonic responsive microbubble production

1. 리피드 쉘 물질 준비1. Lipid Shell Material Preparation

Normal saline + Glycerol + Propylene glycol이 혼합된 용매에 DPPC + DPPA 파우더를 넣고 핫 플레이트(hot plate)를 이용하여 용액이 끓어서 넘치지 않도록 주의하여 3시간 동안 가열한다. 이때, Lipid를 용해할 용매는 Saline, glycerol, propylene glycol을 20:1:21의 비율로 혼합하여 total 100ml의 혼합물에 DPPC(0.1g), DPPA(0.01g)의 비율로 Lipid 파우더를 넣어준다. 이때, DSPE-mPEG(0.127g)를 더 혼합할 수 있다. 그리고, 가열은 microwave를 이용할 수도 있다.Add DPPC + DPPA powder to the solvent mixed with Normal saline + Glycerol + Propylene glycol and heat for 3 hours using a hot plate, taking care not to boil the solution and overflow. At this time, as the solvent for dissolving the lipid, saline, glycerol, and propylene glycol are mixed in a ratio of 20:1:21, and DPPC (0.1g) and DPPA (0.01g) of DPPC (0.1g) and DPPA (0.01g) are added to the total 100ml mixture. At this time, DSPE-mPEG (0.127 g) may be further mixed. And, the heating may use a microwave.

2. 콜레스테롤 쉘 물질 준비2. Preparation of Cholesterol Shell Material

Normal saline + Propylene glycol (+ Glycerol, 생략가능)을 유리 비커에 넣고 전체 100ml의 혼합물에 콜레스테롤(0.127g)을 넣은 뒤 핫 플레이트를 이용하여 용액의 온도를 80°C로 유지하며 3시간 동안 가열한다.Put Normal saline + Propylene glycol (+ Glycerol, optional) in a glass beaker, add cholesterol (0.127 g) to the total mixture of 100 ml, and heat for 3 hours while maintaining the temperature of the solution at 80 °C using a hot plate. .

3. 초음파 반응형 미소기포 제작3. Ultrasonic responsive microbubble production

준비된 DPPC+DPPA 혼합액(1.5ml)과 Cholesterol 혼합액(0.5ml)을 더해준 뒤, perfluorobutane(0.1ml)을 쉘 물질의 솔루션과 코어 가스의 혼합비율(v/v)을 20:1 비율로 혼합하여 2ml 바이얼에 분주하여 밀봉 후 45초 동안 기계적 믹싱한다. 이때, 믹싱은 1분에 4530±100번 진동이 이루어지도록 주파수를 설정한다.After adding the prepared DPPC+DPPA mixed solution (1.5ml) and Cholesterol mixed solution (0.5ml), mix perfluorobutane (0.1ml) with the shell material solution and the core gas mixing ratio (v/v) at a 20:1 ratio to 2ml Dispense into vials and mechanically mix for 45 seconds after sealing. At this time, the mixing frequency is set so that it vibrates 4530±100 times per minute.

즉, 실시예1에서는 초음파 반응형 미소기포(11) 제조의 일례로 DPPC(0.1g) + DPPA(0.01g) + Cholesterol(0.127g)를 각각 용매에 녹여 혼합하였으며, 주입된 가스의 양은 perfluorobutane의 경우 액화상태(-80℃~-20℃)에서 10~100ul (질량으로 환산 시 17.5~175 mg) 이다.That is, in Example 1, each of DPPC (0.1 g) + DPPA (0.01 g) + Cholesterol (0.127 g) was dissolved in a solvent and mixed as an example of the production of ultrasonically responsive microbubbles 11, and the amount of injected gas was the amount of perfluorobutane. In case of liquefied state (-80℃~-20℃), it is 10~100ul (17.5~175 mg in terms of mass).

4. 초음파 반응형 미소기포의 사이즈 조절 및 분리4. Size adjustment and separation of ultrasonic responsive microbubbles

다양한 크기로 제작된 초음파 반응형 미소기포(11)를 폴리카보나이트 멤브레인 필터와 압출기를 사용하여 필터링하고, 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)로 워싱하여 초음파 반응형 미소기포(11)를 완성한다.Ultrasonic responsive microbubbles 11 manufactured in various sizes are filtered using a polycarbonite membrane filter and an extruder, the micelles well composed by centrifugation are settled by centrifugation through a centrifuge, and then the supernatant present in the upper layer is removed and DW (deionized water) to complete the ultrasonic responsive microbubbles 11.

5. 공초점 현미경 분석5. Confocal Microscopy Analysis

상기 실시예1에서와 같은 방법에 의해 NBD PC로 제작된 미셀을 이용하여 미셀의 형성 여부와 형태 분석 및 형광 지질의 위치와 물리적 기포의 상관관계를 확인하기 위하여 도 3에서와 같은 공초점 현미경 영상을 획득하였다.Confocal microscopy image as shown in FIG. 3 in order to check whether micelles are formed and morphologically analyzed using micelles made of NBD PC by the same method as in Example 1, and the correlation between the location of fluorescent lipids and physical bubbles. was obtained.

도 3에서 (a)는 형광 현미경 이미지이며, (b)는 광학 현미경 이미지이며, (c)는 형광 현미경 이미지와 광학 현미경 이미지를 합한(merging) 이미지이다.In FIG. 3, (a) is a fluorescence microscope image, (b) is an optical microscope image, and (c) is a merging image of a fluorescence microscope image and an optical microscope image.

도 3의 (a)와 (b)를 통해 알 수 있는 바와 같이, 형광 신호를 띄는 지질이 쉘 형태로 미셀 테두리를 형성하고 있는 것을 확인할 수 있으며, 가운데 코어 부분은 지질이 아닌 빈 공간 혹은 가스로 이루어져 있는 것을 확인할 수 있다.As can be seen from (a) and (b) of Figure 3, it can be seen that the lipids displaying the fluorescent signal form the micellar border in the form of a shell, and the central core part is not a lipid but an empty space or gas. You can check what is made up.

그리고, 도 3의 (c)를 통해 알 수 있는 바와 같이, 일반 공기방울과 미셀의 구분은 형광지질로 이루어져 있지 않은 기포와 형광 지질로 구성된 기포로 구분할 수 있으며, 대부분의 경우 형광 지질로 쉘이 구성되어 있음을 알 수 있다.And, as can be seen from (c) of FIG. 3, the division of normal air bubbles and micelles can be divided into bubbles not composed of fluorescent lipids and bubbles composed of fluorescent lipids, and in most cases, the shell is composed of fluorescent lipids. It can be seen that it is composed of

6. 입도분석6. Particle size analysis

입도분석의 원리는 레이저가 샘플을 통과할 때 발생하는 회절, 산란광을 이용하여 입자의 크기를 측정하는 것으로, 미셀의 입도분석에 사용된 장비 스펙은 다음과 같다.The principle of particle size analysis is to measure the particle size using diffraction and scattered light generated when a laser passes through a sample. The equipment specifications used for particle size analysis of micelles are as follows.

가. 모델명: ELS-2000ZSgo. Model Name: ELS-2000ZS

나. 입도분석: DLS (Dynamic Light Scattering)me. Particle size analysis: DLS (Dynamic Light Scattering)

다. 제타전위: ELS (Electrophoretic Light Scattering)all. Zeta Potential: ELS (Electrophoretic Light Scattering)

라. 모든 용매에 분산된　Particle의 입도분포 및　Zeta Potential　측정la. Measurement of particle size distribution and Zeta Potential of particles dispersed in all solvents

마. Flat Sample의 표면　Zeta Potential　측정 가능mind. Surface 　 Zeta Potential 　 of flat samples can be measured

바. 온도　Control &　경시변화 측정 가능bar. Temperature 　 Control & 　 Time-lapse measurement possible

사. 미량　Sample　측정 가능buy. Trace 　 Sample 　 Measurable

아. Size: 0.1nm~10000nm / Zeta Potential:　1nm~50000nm　대응 가능Ah. Size: 0.1nm~10000nm / Zeta Potential:　1nm~50000nm　Available

자. 시료 농도: 0.001~40% 대응 가능ruler. Sample concentration: 0.001 to 40% available

도 4의 (a)는 미셀에 대한 입도분석 결과를 Intensity distribution, Volume distribution, Number distribution으로 나타낸 것이다.Figure 4 (a) shows the results of particle size analysis for micelles as Intensity distribution, Volume distribution, and Number distribution.

그 결과 Intensity distribution: 308 nm, Volume distribution: 184 nm, Number Distribution: 139nm로 최종 결과치가 나타난다.As a result, the final result is shown as Intensity distribution: 308 nm, Volume distribution: 184 nm, and Number Distribution: 139 nm.

그리고, 도 4의 (b)는 실시예1에 따라 제작된 미셀의 직경과 분산도 및 확산 상수, 측정환경 등에 대한 경과를 나타낸 것이다. 이때, 기포의 크기 분포 측정 환경은 25℃의 물에서 측정을 하였으며 점도 0.8878(cP), 분산 강도 25762(cps), 감쇠기 0.72(%)로 설정하였다.And, Figure 4 (b) shows the progress of the diameter, dispersion, diffusion constant, measurement environment, etc. of the micelles prepared according to Example 1. At this time, the bubble size distribution measurement environment was measured in water at 25° C., and the viscosity was 0.8878 (cP), the dispersion strength 25762 (cps), and the attenuator was set to 0.72 (%).

측정 결과, 실시예1에 의해 제작된 미셀의 평균 직경은 257.1 nm이며, 확산상수는 1.913 e-8(cm2/sec)로 나타났다.As a result of the measurement, the average diameter of the micelles prepared in Example 1 was 257.1 nm, and the diffusion constant was 1.913 e-8 (cm 2 /sec).

다음으로는, 상기에서와 같은 방법에 의해 만들어진 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)를 이용하여 초음파 반응형 미소기포(11)와 약물을 포함하는 리포좀을 제조하는 방법을 설명하면 다음과 같다.Next, a method of preparing liposomes containing the ultrasonically responsive microbubbles 11 and a drug using the ultrasonically responsive microbubbles 11 having a uniform size distribution made by the method as described above will be described. As follows.

먼저, 리포좀을 제조하기 위한 제2쉘 물질의 솔루션을 준비한다.First, a solution of the second shell material for preparing liposomes is prepared.

이를 위해 제2 리피드를 포함하는 제2 혼합물 파우더를 유기 용매에 용해한 후 유기 용매를 제거하여 리피드 필름을 획득하는 과정을 수행할 수 있다. 여기서 제2 리피드가 포함된 제2 혼합물 파우더에는 알부민(albumin), 폴리머(polymer) 및 PEG(polyethylene glycol) 중 적어도 하나 이상이 더 포함될 수 있으며, 리포좀의 내구성을 높여주기 위해 콜레스테롤이 첨가될 수 있다.To this end, a process of obtaining a lipid film by dissolving a second mixture powder including a second lipid in an organic solvent and then removing the organic solvent may be performed. Here, the second mixture powder including the second lipid may further include at least one of albumin, polymer, and PEG (polyethylene glycol), and cholesterol may be added to increase the durability of the liposome. .

또한, 제2 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg 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(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함할 수 있다.In addition, the second lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3) -phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA ( Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2- Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Diolephosphatidylethanolamine) -glycerol -3-phosphoethanolamine), egg 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 (ioleoyl phosphatidylserine), DMPS (Dimyristoyl phosphatidylserine), DMPS-Na (1,2-Dimyristoyl-sn-glycerol) -3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE -mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3- phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, and at least one of dipotassium glycyrrhizinate may be included.

일 예로, 약물이나 유전자를 포집하기 위한 미셀은 약물이나 유전자를 채집하고 미셀의 안정성을 높이기 위해 쉘 물질로 DPPC (Dipalmitoyl -Phos Phatidyl - Choline), DPPA(Diphenyl-phosphoryl-Azide)를 주요 구성물로 할 수 있다. 이때, DPPC, DPPA, 및 콜레스테롤은 60%~85%:2%~10%:10%~30%의 비율로 혼합될 수 있다. 그리고, 쉘 물질로 DSPE-mPEG가 추가될 수 있다.For example, in micelles for collecting drugs or genes, DPPC (Dipalmitoyl -Phos Phatidyl - Choline) and DPPA (Diphenyl-phosphoryl-Azide) as the main components are used as shell materials to collect drugs or genes and increase the stability of micelles. can At this time, DPPC, DPPA, and cholesterol may be mixed in a ratio of 60% to 85%: 2% to 10%: 10% to 30%. And, DSPE-mPEG may be added as a shell material.

특히, DPPA, DMPA-Na, DPPA-Na, DSPG, DSPS 등은 음전하 또는 양전하를 가지는 인지질로 리포좀이 전하를 띄게 하기 위하여 사용되어질 수 있다.In particular, DPPA, DMPA-Na, DPPA-Na, DSPG, DSPS, etc. can be used to charge the liposome with a phospholipid having a negative or positive charge.

또한, 알부민은 세럼 알부민 (serum albumin), 오발부민 (ovalbumin) 등을 포함할 수 있다.In addition, the albumin may include serum albumin, ovalbumin, and the like.

또한, 폴리머는 Poly(β-benzyl-L-asparate), PBLA(poly-DL-lactic acid) 등을 포함할 수 있다.In addition, the polymer may include poly(β-benzyl-L-asparate), poly-DL-lactic acid (PBLA), and the like.

여기서, 유기용매는 클로로포름과 메탄올의 혼합 용매를 포함할 수 있으며, 클로로포름과 메탈올은 1:1 내지 3:1의 혼합비로 혼합될 수 있다.Here, the organic solvent may include a mixed solvent of chloroform and methanol, and chloroform and metalol may be mixed in a mixing ratio of 1:1 to 3:1.

또한, 제2 혼합물 파우더를 유기 용매에 녹이기 위하여 자석 교반기(magnetic stirrer)를 사용하여 교반할 수 있으며, 교반기의 온도는 약 40°C~60°C로 하여 10분 내지 30분간 교반을 진행할 수 있다.In addition, in order to dissolve the second mixture powder in the organic solvent, it can be stirred using a magnetic stirrer, and the temperature of the stirrer is set to about 40 °C to 60 °C, and stirring can be performed for 10 to 30 minutes. .

또한, 회전 증발 방식에 의해 쉘 물질이 녹아있는 유기 용매를 제거할 수 있다.In addition, the organic solvent in which the shell material is dissolved may be removed by rotary evaporation.

일 예로, 회전증발기(rotary evaporator)를 이용하여 20°C 내지 40°C의 온도에서 10분 내지 30분간 유기 용매를 증발시키며, 잔류된 유기 용매를 완전히 제거하기 위하여 진공 챔버에 넣은 후 감압 건조하여 준다. 이때 진공 챔버에서의 감압 건조는 적어도 6시간 이상 진행하는 것이 바람직하며, 보다 상세하게는 24시간 진행하는 것이 바람직하다.For example, by using a rotary evaporator (rotary evaporator) to evaporate the organic solvent at a temperature of 20 °C to 40 °C for 10 to 30 minutes, and then put in a vacuum chamber to completely remove the remaining organic solvent and then dried under reduced pressure. give. At this time, the drying under reduced pressure in the vacuum chamber is preferably carried out for at least 6 hours, and more specifically, it is preferable to proceed for 24 hours.

그러면, 유기 용매의 클로로포름과 메탄올 등이 증발된 뒤 플라스크 바닥에는 리피드 필름이 다층으로 형성될 수 있으며, 플라스크 바닥에는 뿌옇게 리피드 물질이 회전이 적용된 형태로 필름 케이크(film cake)를 형성하게 된다.Then, after chloroform and methanol of the organic solvent are evaporated, a lipid film may be formed in multiple layers on the bottom of the flask, and a film cake is formed in the bottom of the flask in which the lipid material is turned cloudy.

다음으로, 리피드 필름에 적절한 용량의 제2 용매, 일 예로, PBS(Phosphate-buffered saline)를 넣어 수화함으로써 제2쉘 물질의 솔루션을 생성한 다음, 초음파발생장치를 이용하여 분쇄하여 준다. 이때, 약물의 조건 및 미셀의 특성에 따라 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 함께 넣고 초음파 분해를 할 수 있으나 이에 한정되는 것은 아니며 리피드 필름을 분쇄한 이후에 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 넣을 수 있다.Next, a second solvent of an appropriate capacity, for example, PBS (Phosphate-buffered saline) is added to the lipid film to produce a solution of the second shell material and then pulverized using an ultrasonicator. At this time, according to the conditions of the drug and the characteristics of the micelles, the ultrasonically responsive microbubbles 11 having a uniform size distribution and the drug and/or gene may be put together and subjected to sonication, but the present invention is not limited thereto. Thereafter, the ultrasound-responsive microbubbles 11 and drugs and/or genes may be added.

그러면, 초음파발생장치에 의해 분쇄된 리피드 필름과 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)와 약물 및/또는 유전자는 자기정합(self-assembling) 기전에 의해 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 포함하는 리포좀의 형태로 제작된다.Then, the lipid film pulverized by the ultrasonic generator and the ultrasonically responsive microbubbles 11 with uniform size distribution and the drug and/or gene are self-assembling by the ultrasonically responsive microbubbles 11 ) and is produced in the form of liposomes containing drugs and/or genes.

다음으로, 도 5에서와 같이, 다양한 크기로 제작된 리포좀을 필터와 압출기를 사용하여 필터링하여 사이즈를 조절한다. 즉, 필터의 공극보다 큰 직경을 가지는 리포좀은 공극을 통과하지 못하고 파괴되며, 파괴된 리포좀 내부에 위치하는 초음파 반응형 미소기포(11)와 약물, 리피드 필름은 재결합하게 된다.Next, as shown in FIG. 5, the liposomes manufactured in various sizes are filtered using a filter and an extruder to adjust the size. That is, the liposome having a larger diameter than the pores of the filter is destroyed without passing through the pores, and the ultrasonically responsive microbubbles 11 located inside the destroyed liposomes, the drug, and the lipid film are recombined.

이후, 필터링에 의해 크기 분포가 균일하게 형성된 리포좀을 포함하는 혼합물을 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)를 이용하여 워싱함으로써 원하는 크기로 조정된 리포좀을 획득할 수 있다.Thereafter, the mixture containing the liposomes having a uniform size distribution by filtering sinks the micelles well-formed by centrifugation through a centrifuge, and then the supernatant present in the upper layer is removed and washed using DW (deionized water). Liposomes adjusted to a desired size can be obtained.

실시예2. 리포좀 제작Example 2. Liposome production

1. 쉘 물질 준비1. Shell Material Preparation

DPPC + DPPA + Cholesterol을 각각 75:5:20 비율로 혼합하여 리피드 물질을 생성하고, 생성된 리피드 물질을 클로로포름/메탄올(2:1, v/v)의 유기 용매에 녹인다.DPPC + DPPA + Cholesterol are each mixed in a ratio of 75:5:20 to produce a lipid material, and the resulting lipid material is dissolved in an organic solvent of chloroform/methanol (2:1, v/v).

그리고, 회전교반기를 이용하여 30℃에서 20분간 유기 용매를 증발시킨다.Then, the organic solvent is evaporated at 30° C. for 20 minutes using a rotary stirrer.

이후, 진공 챔버에 넣은 뒤 감압하여 24시간 감압 건조하여 리피드 필름을 제작한다.Thereafter, it was put in a vacuum chamber and dried under reduced pressure for 24 hours to prepare a lipid film.

2. 리포좀 제작2. Liposome production

리피드 필름에 2ml의 PBS를 넣어 수화한 다음, 상온에서 1분 동안 100W의 에너지로 초음파 분해를 진행한다. 이때, 실시예1에서 제작된 사이즈가 조절된 미소기포(11)와 약물 및/또는 유전자를 함께 넣고 초음파 분해를 진행하여 다양한 크기를 가지는 리포좀을 제작할 수 있다.After hydration by putting 2ml of PBS in the lipid film, ultrasonication is performed at 100W energy for 1 minute at room temperature. At this time, the size-controlled microbubbles 11 prepared in Example 1 and the drug and/or gene are put together and sonicated to produce liposomes having various sizes.

3. 리포좀의 사이즈 조절 및 재결합3. Liposome size control and recombination

다양한 크기로 제작된 초음파 반응형 미소기포(11)를 폴리카보나이트 멤브레인 필터와 압출기를 사용하여 필터링하고, 원심분리기를 통해 잘 구성된 리포좀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)로 워싱하여 약물전달을 위한 리포좀을 완성한다Ultrasonic responsive microbubbles 11 manufactured in various sizes are filtered using a polycarbonate membrane filter and an extruder, the well-formed liposomes are settled through a centrifuge, and the supernatant present in the upper layer is removed and deionized water (DW) ) to complete the liposome for drug delivery

4. 공초점 현미경 분석4. Confocal Microscopy Analysis

상기 실시예2에서와 같은 방법에 의해 약물을 포집하는 리포좀을 공초점 현미경을 통해 관찰한 결과 도 6에서 알 수 있는 바와 같이, 내부에 가스 공간이 존재하는 미소기포(11)의 포집 및 미셀 외부에 친수성 형광 물질(MW: 4k의 Dextran)이 포집된 리포좀이 확인됨을 알 수 있다.As can be seen from FIG. 6 as a result of observing the liposome collecting the drug by the same method as in Example 2 through a confocal microscope, the collection of microbubbles 11 having a gas space therein and the outside of the micelles It can be seen that the liposome in which the hydrophilic fluorescent substance (MW: 4k of Dextran) is captured is confirmed.

또한, 도 7에서 알 수 있는 바와 같이, 초음파 반응형 미소기포(11)와 리포좀의 사이즈를 조절하지 않은 상태인 (a)에 비하여, 본 발명에서의 실시예에 따라 제작된 상태인 (b)에서 각각의 리포좀들이 효율적으로 약물을 포집하고 있음을 확인할 수 있다.In addition, as can be seen from FIG. 7 , compared to (a) in which the size of the ultrasonically responsive microbubbles 11 and the liposomes are not adjusted, (b) in a state prepared according to an embodiment of the present invention It can be confirmed that each liposome efficiently traps the drug.

5. 골드 나노 파티클 포집 실험5. Gold Nanoparticle Capture Experiment

TEM 영상을 이용하여 초음파 반응형 미소기포(11)와 친수성 약물을 함유한 리포좀을 분석하기 위한 것으로, 도 8의 (a)와 같이 골드 나노 파티클이 위치하는 영역에 본 발명의 일 실시예에 따라 제작된 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀을 위치시켰다.To analyze the liposome containing the ultrasonically responsive microbubbles 11 and the hydrophilic drug using a TEM image, as shown in FIG. The prepared ultrasonically responsive microbubbles 11 and the drug-containing liposomes were placed.

그리고, TEM 영상을 통해 상태를 확인한 결과, 도 8의 (b)에서와 같이 골드 나노 파티클이 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀에 포획되는 것을 알 수 있으며, 이를 통해 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀에 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물이 탑재 되었음을 확인할 수 있다.And, as a result of confirming the state through the TEM image, it can be seen that the gold nanoparticles are captured in the liposome containing the ultrasonically responsive microbubbles 11 and the drug, as shown in FIG. It can be confirmed that a certain amount or more of the drug is loaded to exhibit a significant drug effect on the liposome containing the microbubbles 11 and the drug.

상기에서 제작된 약물전달을 위한 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀은 특정 영역에서 초음파 에너지를 받게 되면 캐비테이션 효과를 발생시켜 리포좀에 함유된 약물이 해당 위치에서 방출될 수 있도록 할 수 있다.The liposome containing the ultrasonically responsive microbubbles 11 and drug for drug delivery produced above generates a cavitation effect when receiving ultrasonic energy in a specific area so that the drug contained in the liposome can be released at the corresponding position. can

또한, 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀은 표적 전달 효율을 높이기 위해 암세포와 같은 특정 세포 표면에서 발현하는 단백질 등의 표적 분자에 반응하도록 만들어진 항체나 펩타이드와 같은 타게팅 리간드(targeting ligand)를 리포좀 표면에 결합할 수도 있다.In addition, the ultrasonically responsive microbubbles 11 and drug-containing liposomes for drug delivery according to an embodiment of the present invention target molecules such as proteins expressed on the surface of specific cells, such as cancer cells, in order to increase target delivery efficiency. A targeting ligand such as an antibody or peptide made to react may be bound to the liposome surface.

즉, 본 발명의 일 실시예에 따른 리포좀은 포집된 약물을 이용하는 집속형 초음파에 의한 수동형 표적 지향과 특정 리간드에 의한 능동적 표적 지향의 표적 효과를 동시에 가져올 수 있다.That is, the liposome according to an embodiment of the present invention can bring about the target effect of passive targeting by focused ultrasound using the captured drug and active targeting by a specific ligand at the same time.

이때, 표적하는 병원체에 대한 리간드 라이브러리 스크리닝을 통해 항체, 단백질, 펩타이드, 수용체 등의 표적 대상을 확인하고, 확인된 표적 대상을 리포좀 표면에 결합한다. 그러면, 리포좀 표면에 결합된 표적 대상이 병원체에 유도될 수 있게 된다.In this case, a target target such as an antibody, protein, peptide, or receptor is identified through ligand library screening for a target pathogen, and the identified target target is bound to the liposome surface. Then, the target bound to the liposome surface can be induced to the pathogen.

또한, 리포좀 표면에 표적 대상을 결합하는 방법은, 예를 들어, 상기 실시예1 및 실시예2에 의해 PEG-COOH가 결합되어있는 리포좀을 만든다(DNase, RNase free water).In addition, the method of binding the target to the liposome surface, for example, according to Examples 1 and 2 above, makes liposomes to which PEG-COOH is bound (DNase, RNase free water).

그리고, EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), N-hydroxysuccinimide를 첨가하여 15분간 실온에 교반시켜 카르복실기(COOH)가 활성화되도록 해준다.Then, EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and N-hydroxysuccinimide are added and stirred at room temperature for 15 minutes to activate the carboxyl group (COOH).

다음으로 N-hydroxysuccinimide가 활성화된 리포좀에 아미노기(NH2)가 결합되어있는 표적 대상을 넣어 카르복실과 아민이 반응하여 아마이드가 형성되어 결합되도록 해준다.Next, a target target having an amino group (NH2) is added to the N-hydroxysuccinimide-activated liposome so that the carboxyl and amine react to form an amide and bind it.

이상에서 본 발명이 구체적인 구성요소 등과 같은 특정 사항들과 한정된 실시예 및 도면에 의해 설명되었으나, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐, 본 발명이 상기 실시예들에 한정되는 것은 아니며, 본 발명이 속하는 기술분야에서 통상적인 지식을 가진 자라면 이러한 기재로부터 다양한 수정 및 변형을 꾀할 수 있다.In the above, the present invention has been described with specific matters such as specific components and limited embodiments and drawings, but these are provided to help a more general understanding of the present invention, and the present invention is not limited to the above embodiments. , various modifications and variations can be devised from these descriptions by those of ordinary skill in the art to which the present invention pertains.

따라서, 본 발명의 사상은 상기 설명된 실시예에 국한되어 정해져서는 아니 되며, 후술하는 특허청구범위뿐만 아니라 이 특허청구범위와 균등하게 또는 등가적으로 변형된 모든 것들은 본 발명의 사상의 범주에 속한다고 할 것이다.Therefore, the spirit of the present invention should not be limited to the above-described embodiments, and not only the claims described below but also all modifications equivalently or equivalently to the claims described below belong to the scope of the spirit of the present invention. will do it

Claims (2)

약물전달을 위한 리포좀에 있어서,
불활성 가스를 내부에 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포;
상기 초음파 반응형 미소기포 및 약물을 수용하기 위한 제2쉘;
을 포함하되,
상기 초음파 반응형 미소기포의 크기 분포가 제1 균일 크기 분포이고,
상기 리포좀의 크기 분포가 제2 균일 크기 분포이며,
상기 제1쉘 및 상기 제2쉘 각각은, DPPA(Diphenylphosphoryl azide), DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine) 및 콜레스테롤을 포함하되,
상기 제1쉘의 상기 DPPA, 상기 DPPC 및 상기 콜레스테롤의 질량비는 10:100:127이며,
상기 제2쉘의 상기 DPPA, 상기 DPPC 및 상기 콜레스테롤의 질량비는 5:75:20이고,
상기 초음파 반응형 미소기포가 생성된 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포가 상기 제1 균일 크기 분포가 되도록 상기 초음파 반응형 미소기포의 크기가 조절되며,
상기 제1 균일 크기 분포를 가지는 상기 초음파 반응형 미소기포 및 상기 약물을 내부에 포함하는 상기 리포좀이 생성된 이후 상기 압출기를 통해 상기 리포좀의 크기 분포가 상기 제2 균일 크기 분포가 되도록 상기 리포좀의 크기가 조절되고,
상기 초음파 반응형 미소기포의 상기 제1 균일 크기 분포는 상기 리포좀의 상기 제2 균일 크기 분포보다 작은 것을 특징으로 하는 리포좀.
In the liposome for drug delivery,
Ultrasonic responsive microbubbles containing an inert gas inside and having a first shell formed on an outer surface thereof;
a second shell for accommodating the ultrasonically responsive microbubbles and drugs;
including,
The size distribution of the ultrasonically responsive microbubbles is a first uniform size distribution,
The size distribution of the liposome is a second uniform size distribution,
Each of the first shell and the second shell includes DPPA (Diphenylphosphoryl azide), DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine) and cholesterol,
The mass ratio of the DPPA, the DPPC, and the cholesterol of the first shell is 10:100:127,
The mass ratio of the DPPA, the DPPC and the cholesterol of the second shell is 5:75:20,
After the ultrasonic responsive microbubbles are generated, the size of the ultrasonically responsive microbubbles is adjusted through an extruder so that the size distribution of the ultrasonically responsive microbubbles becomes the first uniform size distribution,
The size of the liposome so that the size distribution of the liposome becomes the second uniform size distribution through the extruder after the liposome including the ultrasonically responsive microbubbles having the first uniform size distribution and the drug therein is generated is regulated,
The first uniform size distribution of the ultrasonically responsive microbubbles is smaller than the second uniform size distribution of the liposome.
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