KR101487088B1 - Ultrasound contrast agent with nanoparticles including drug and method for preparing the same - Google Patents
Ultrasound contrast agent with nanoparticles including drug and method for preparing the same Download PDFInfo
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- KR101487088B1 KR101487088B1 KR20130070039A KR20130070039A KR101487088B1 KR 101487088 B1 KR101487088 B1 KR 101487088B1 KR 20130070039 A KR20130070039 A KR 20130070039A KR 20130070039 A KR20130070039 A KR 20130070039A KR 101487088 B1 KR101487088 B1 KR 101487088B1
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- KR
- South Korea
- Prior art keywords
- nanoparticles
- microbubble
- contrast agent
- ultrasound contrast
- drug
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Abstract
본 발명은 약물 수송능력을 증가시키고 약물 침투효과를 높인 나노입자가 결합된 초음파 조영제 및 이의 제조방법에 관한 것이다.
본 발명에 따른 초음파 조영제는 약물을 적재한 나노입자가 표면에 결합되어 있어 약물의 수송능력 및 초음파에 의한 나노입자의 침투능력이 향상되었다. 또한, 본 발명은 암에 대해 표적 지향적 특성을 가지는 단백질을 활용하여 나노입자를 제조함에 따라 암의 선택적 진단 및 치료를 동시에 수행할 수 있다. The present invention relates to a nanoparticle-coupled ultrasound contrast agent that increases drug transport capability and enhances drug penetration, and a method for producing the same.
The ultrasound contrast agent according to the present invention has nanoparticles loaded with a drug bound to the surface thereof, thereby improving drug transport ability and penetration ability of nanoparticles by ultrasonic waves. In addition, the present invention can simultaneously perform selective diagnosis and treatment of cancer by preparing nanoparticles using a protein having a target-directed property to cancer.
Description
본 발명은 약물을 함유한 나노입자가 결합된 초음파 조영제 및 이의 제조방법에 관한 것으로서, 보다 자세하게는, 본 발명은 약물 수송능력을 증가시키고 약물 침투효과를 높인 나노입자가 결합된 초음파 조영제 및 이의 제조방법에 관한 것이다.The present invention relates to a nanoparticle coupled ultrasound contrast agent and a method for preparing the same. More particularly, the present invention relates to a nanoparticle-coupled ultrasound contrast agent that increases the drug transport capability and enhances drug penetration, ≪ / RTI >
초음파 영상장치는 실시간으로 진단이 가능하여 빠른 결과를 도출할 수 있는 최고의 장점을 가지고 있으며 MRI, CT와는 달리 구조가 간단하고 비용이 저렴하다. 초음파 조영제를 투여한 다음 초음파를 가하면 초음파가 조영제내의 미세기포에 의해 반사되어 내부 장기의 영상을 더욱 뚜렷하게 보여준다. 이러한 초음파 조영제는 1968년에 Gramiak과 Shah가 혈관 내에 미세 기포 (microbubble)를 주입한 후 초음파 신호가 증강되는 것을 발견한 것을 모태로 하여 발전하였다. 지금까지 알려진 조영제로는 미국특허 제 4,276,885호의 젤라틴 외피로 둘러싼 소형 기포, 미국특허 제 4,265,251호의 다당류 고체 주변부 벽을 갖는 소형기체, 유럽특허공보 제 52575호의 고체 결정상 화합물의 미립자(예: 갈라토즈)를 이용한 소형기포, 유럽특허 공개 제 0122624호의 지방산을 이용한 소형기포 및 대한민국 특허 제 1989-2989호의 지방산 및 계면활성제를 이용하여 제조한 소형기포가 있으며, 동맥혈에서도 조영효과를 나타내는 시판 조영제로는 쉐링사(Shering)의 레보비스트(Levovist)와 미국 말린크로드트(Mallinckrodt)사의 알부넥스(Albunex), 옵티슨(Optison) 등이 있다. The ultrasound imaging device has the best advantage of being able to diagnose in real time and to obtain fast results. Unlike MRI and CT, it is simple in structure and low cost. When the ultrasound is applied after applying the ultrasound contrast agent, the ultrasound is reflected by the minute bubbles in the contrast agent, and the image of the internal organs is more clearly displayed. These ultrasound contrast agents were developed on the basis of the finding that in 1968, Gramiak and Shah injected microbubbles into the blood vessels to enhance the ultrasound signals. Contrast agents known so far include small bubbles surrounded by a gelatin shell of US Pat. No. 4,276,885, small gases having polysaccharide solid perimeter walls of US Pat. No. 4,265,251, microparticles of a solid crystalline compound of European Patent Publication No. 52575 (eg Galatose) Small bubbles using fatty acids of European Patent Publication No. 0122624 and small bubbles produced using fatty acids and surfactants of Korean Patent No. 1989-2989 and commercial contrast agents showing arterial blood vessels are commercially available from Schering Co. Levovist of Shering and Albinex and Optison of Mallinckrodt of the United States.
한편, 의료 기술 및 치료의 기술의 발달에 따라서 현재의 진단과 치료를 동시에 진행하기 위한 테라그노시스(theragnosis)기술의 개발이 활발하게 연구되고 있다. 이러한 측면에서 초음파 조영제를 담체로 약물을 전달함으로써 진단과 치료를 동시에 할 수 있는 초음파 조영제의 개발이 시도되고 있다. 하지만 일반적으로 초음파 진단을 위하여 사용되는 초음파 조영제의 구조는 약물을 담을 수 있는 공간이 매우 협소하여 충분한 약물을 적재하기 힘들어 치료의 효과를 비약적으로 상승시키기 어려운 문제가 있다. On the other hand, development of theragnosis technology for simultaneously proceeding diagnosis and treatment according to development of medical technology and treatment technology is actively studied. In this respect, development of an ultrasound contrast agent capable of simultaneous diagnosis and treatment by delivering a drug to a carrier using an ultrasound contrast agent has been attempted. However, in general, the structure of the ultrasound contrast agent used for ultrasound diagnosis has a problem that it is difficult to increase the effect of the treatment because the space for holding the drug is very narrow and it is difficult to load sufficient drugs.
본 발명은 약물의 수송 및 초음파에 의한 약물의 침투 능력을 높여 진단 및 치료를 동시에 수행할 수 있는 초음파 조영제를 제공하는 것이다.The present invention provides an ultrasound contrast agent capable of simultaneously carrying out diagnosis and treatment by increasing drug penetration ability by drug transport and ultrasound.
본 발명은 세포내로의 침투효과를 극대화하고 약물을 지속적으로 방출할 수 있는 기능성 초음파 조영제를 제공하는 것이다.The present invention provides a functional ultrasound contrast agent capable of maximizing penetration into a cell and releasing a drug continuously.
본 발명의 하나의 양상은 내부에 가스가 충전된 마이크로버블 ; 상기 마이크로 버블의 표면에 약물을 함유하는 나노입자가 결합된 초음파 조영제에 관계한다.One aspect of the present invention relates to a micro bubble filled with gas therein; And an ultrasound contrast agent in which nanoparticles containing a drug are bound to the surface of the microbubble.
다른 양상에서 본 발명은 마이크로버블과 약물을 함유하는 나노입자를 각각 제조하는 단계 ; 상기 나노입자와 상기 마이크로버블을 소정 비율로 물에 혼합하여 반응시키는 단계를 포함하는 초음파 조영제의 제조방법에 관계한다.In another aspect, the present invention provides a method of preparing a nanoparticle comprising: preparing a microbubble and a drug-containing nanoparticle, respectively; Mixing the nanoparticles with the microbubbles in water at a predetermined ratio, and reacting the nanoparticles with water.
본 발명에 따른 초음파 조영제는 약물을 적재한 나노입자가 표면에 결합되어 있어 약물의 수송능력이 향상되었다. 또한, 본 발명은 암에 대해 표적 지향적 특성을 가지는 단백질을 활용하여 나노입자를 제조함에 따라 암의 선택적 진단 및 치료를 동시에 수행할 수 있으며 초음파의 효과를 이용하여 세포내의 침투효과를 향상시켜 치료의 효과를 극대화하였다. The ultrasound contrast agent according to the present invention has enhanced drug transport ability because nanoparticles loaded with drugs are bonded to the surface. In addition, the present invention can simultaneously perform selective diagnosis and treatment of cancer by preparing nanoparticles using a protein having a target-directed property to cancer, and by using the effect of ultrasound to enhance intracellular penetration, Maximizing the effect.
도 1은 본 발명의 일구현예에 의한 초음파 조영제의 개념도를 나타낸다.
도 2는 본 발명의 초음파 조영제 제조 프로세스를 보여준다.
도 3은 본 발명의 마이크로 버블, 약물을 함유한 Human Serum Albumin(HSA) 나노입자 및 이들이 결합된 초음파 조영제의 confocal fluorescence microscope 이미지이다.
도 4는 본 발명의 HSA 나노입자가 결합된 초음파 조영제의 크기 분포를 나타낸다.
도 5는 실시예 1과 대조군인 water를 agarose 팬텀에 넣고 초음파 진단을 위한 상용화 기기를 이용하여 하모닉 모드로 초음파 영상을 확인한 결과이다.
도 6은 초음파 조영제에 의하여 반사/투과된 초음파를 하이드로폰을 이용하여 초음파 성분을 수신, 이 때 수신된 성분 중 Harmonic 성분만을 검출하여 분석한 그래프이다.
도 7은 3MHz의 초음파를 실시예 1의 HSA-NPs가 결합된 microbubble에 방출하여 수신한 피크(왼쪽)와 물에 방출하여 수신한 피크(오른쪽)과 비교한 그래프이다.
도 8은 비교예 1(HSA-NPs가 결합되지 않은 microbubble)과 실시예 1에 대하여 장시간 초음파를 가했을 때의 초음파 조영제의 안정성을 측정한 결과를 나타낸다.
도 9는 항암제 paclitaxel을 함유하는 HSA-NPs를 이용하여 세포 수준에서의 항암효과를 검증하는 시험을 수행한 그래프이다.
도 10은 실시예 1과 비교예 1의 초음파 영상효과를 Balb/C nude mouse에 MCF-7을 이식한 질병 동물 모델을 이용하여 검증한 영상이다.
도 11은 도 10의 영상처리 결과에 대하여 암에서의 intensity를 Matlab 프로그램을 이용하여 정량 비교한 그래프이다.
도 12는 MCF-7 세포를 Balb/C nude mouse에 이식하여 동물 질병 모델을 제작하여 0.5mm의 크기를 보일 때 실험을 진행하여 mouse의 생존율을 관찰한 것이다. 1 is a conceptual diagram of an ultrasound contrast agent according to an embodiment of the present invention.
2 shows a process for producing an ultrasound contrast agent of the present invention.
FIG. 3 is a confocal fluorescence microscope image of microbubbles, drug-containing Human Serum Albumin (HSA) nanoparticles of the present invention and an ultrasound contrast agent bound thereto.
FIG. 4 shows the size distribution of the ultrasound contrast agent to which the HSA nanoparticles of the present invention are bound.
FIG. 5 is a result obtained by comparing ultrasound images in a harmonic mode using a commercial device for ultrasonic diagnosis by placing water, which is a control group, in Example 1 into an agarose phantom.
FIG. 6 is a graph of ultrasonic waves reflected / transmitted by an ultrasound contrast agent and receiving ultrasonic components using a hydrophone, wherein only the harmonic components of the received components are detected and analyzed.
FIG. 7 is a graph comparing an ultrasound of 3 MHz to a peak (left) emitted from a microbubble coupled with HSA-NPs of Example 1 and a peak received (right) emitted to water.
FIG. 8 shows the results of measuring the stability of an ultrasound contrast agent when Comparative Example 1 (microbubble without HSA-NPs) and Example 1 were subjected to long-term ultrasound.
FIG. 9 is a graph showing a test for verifying the anticancer effect at the cellular level using HSA-NPs containing the anticancer agent paclitaxel.
FIG. 10 is an image obtained by examining the ultrasound imaging effect of Example 1 and Comparative Example 1 using a disease animal model implanted with MCF-7 in a Balb / C nude mouse.
11 is a graph showing a quantitative comparison of intensities in the arm using the Matlab program with respect to the image processing result of FIG.
FIG. 12 shows the survival rate of the mouse when MCF-7 cells were transplanted into a Balb / C nude mouse and an animal disease model was prepared and the size of the animal was 0.5 mm.
본 발명은 하기의 설명에 의하여 모두 달성될 수 있다. 하기의 설명은 본 발명의 바람직한 구체 예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아니다. The present invention can be all accomplished by the following description. The following description should be understood to describe preferred embodiments of the present invention, but the present invention is not necessarily limited thereto.
도 1은 본 발명의 일구현예에 의한 초음파 조영제의 개념도를 나타낸다. 도 1을 참고하면, 본 발명의 초음파 조영제는 마이크로버블(10) 및 나노입자(20)를 포함한다.1 is a conceptual diagram of an ultrasound contrast agent according to an embodiment of the present invention. Referring to FIG. 1, the ultrasound contrast agent of the present invention includes a
상기 마이크로버블(10)은 가스 충전된 마이크로스피어(microspheres), 가스 충전된 리포좀 또는 가스 포밍 에멀젼(gas-forming emulsions)일 수 있다. 바람직하게는 가스 충전된 리포좀을 사용할 수 있다.The
상기 리포좀은 인지질을 포함하는 양친매성 화합물에 의해 형성된다. 이러한 양친매성 화합물은 전형적으로 수성 매질과 본질적으로 수불용성인 유기용매 간의 계면에 배열되어, 유화된 용매 미세방울을 안정화한다. 상기 양친매성 화합물은 수성 매질과 반응할 수 있는 친수성극 머리 부분 (예: 극성 또는 이온성기) 및 예를 들어 유기용매와 반응할 수 있는 소수성 유기 꼬리 부분(예: 탄화수소 사슬)을 가진 분자를 가진 화합물을 포함한다. 양친매성 화합물은 비혼화성인 두 가지의 액체(예: 물과 오일)의 혼합물, 액체와 기체의 혼합물 (예: 물중에 기체 마이크로버블) 또는 액체와 불용성 입자의 혼합물 (예. 물중에 금속 나노 입자)과 같이 다른 방법으로는 통상적으로 섞일 수 없는 물질의 혼합물을 안정화시킬 수 있는 화합물이다. 특히, 본 발명에서는 인지질 박막에 비활성 기체와 물을 주입한 후 초음파 처리하여 내부에 비활성 기체가 충진된 리포좀을 형성한다.The liposome is formed by an amphipathic compound comprising a phospholipid. Such amphipathic compounds are typically arranged at the interface between the aqueous medium and an essentially water-insoluble organic solvent to stabilize the emulsified solvent droplets. The amphipathic compound may have a hydrophilic extreme head portion (e.g., a polar or ionic group) capable of reacting with an aqueous medium and a molecule having a hydrophobic organic tail portion (e.g., a hydrocarbon chain) capable of reacting with, for example, ≪ / RTI > The amphipathic compound may be a mixture of two immiscible liquids (e.g., water and oil), a mixture of liquid and gas (e.g., gas microbubbles in water), or a mixture of liquid and insoluble particles ) Is a compound capable of stabilizing a mixture of substances which are not normally miscible with other methods. Particularly, in the present invention, an inert gas and water are injected into the phospholipid thin film and then subjected to ultrasonic treatment to form a liposome filled with an inert gas therein.
양친매성 인지질 화합물은 적어도 하나의 인산염군을 함유하고, 적어도 하나의, 바람직하게는 두 개의 친유성 긴-사슬 탄화수소기를 함유한다. The amphiphilic phospholipid compound contains at least one phosphate group and contains at least one, preferably two, lipophilic long-chain hydrocarbon groups.
상기 양친매성 인지질로는 공지된 화합물을 사용할 수 있으며, 일예로는 1,2-디스테아르오일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-디아실-sn-글리세로-3-포스포에탄올아민(1,2-diacyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디마이리스토일-sn-글리세로-3-포스파티딜에탄올아민(1,2-Dimyristoyl-snglycero-3-phosphatidylethanolamine, DMPE), 1,2-디스테아르오일-sn-글리세로-3-포스포에탄올아민(1,2- distearoyl-sn-glycero-3-phosphoethanolamine, DSPE) 등이 있다. As the amphiphilic phospholipid, known compounds can be used. For example, 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn- 3-phosphatidylethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2-distearoyl-sn -glycero-3-phosphoethanolamine, DSPE).
또한, 상기 양친매성 인지질 화합물은 변형된 인지질 화합물을 사용할 수 있다. 변형된 인지질의 예는 폴리에틸렌글리콜(PEG)이 첨가된 변형된 인지질의 예는 폴리에틸렌글리콜 (PEG)과 함께 변형된 포스파티딜에탄올아민(DMPE-PEG) 또는 포스포에탄올아민(DSPE-PEG) 등이 있다.In addition, the amphiphilic phospholipid compound may use a modified phospholipid compound. An example of a modified phospholipid is phosphatidylethanolamine (DMPE-PEG) or phosphoethanolamine (DSPE-PEG) which is modified with polyethylene glycol (PEG), and the like, as modified phospholipids to which polyethylene glycol .
바람직하게는, 본 발명에 사용되는 양친매성 인지질 화합물은 아미드 결합을 형성하기 위한 NHS(N-hydroxy succinimide)를 포함할 수 있다. Preferably, the amphiphilic phospholipid compound used in the present invention may comprise an N-hydroxy succinimide (NHS) to form an amide bond.
본 발명에서는 상기 양친매성 화합물 이외에 부가적인 양친매성 물질을 추가로 포함할 수 있는데, 그 일예로는 리소지질, 스테아르산, 폴리에틸렌글리콜, 폴리옥시에틸렌 지방산 에스테르, 폴리옥시에틸렌 지방산 스테아르레이트, 폴리옥시에틸렌 지방 알코올 등이 있을 수 있다.In the present invention, in addition to the amphipathic compound, an additional amphipathic substance may be further included. Examples thereof include lysozyme, stearic acid, polyethylene glycol, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid stearate, polyoxyethylene Fatty alcohol, and the like.
상기 리포좀 내부에 충진되는 가스는 공지된 기체를 제한없이 사용할 수 있으며, 일예로서 이산화탄소, 헬륨, 질소, 아르곤, 설퍼 헥사플루오라이드, 퍼플루오르화 기체를 사용할 수 있다. 상기 기체는 불소가스가 포함된 불화물이 바람직하고, 예로는 퍼플루오로프로판(C3F8), 설퍼 헥사플루오라이드(SF6), 퍼플루오로펜탄(perfluoropentane), 데카플루오로부탄 (decafluorobutane) 및 퍼플루오로헥산(perfluorohexane)가 있다.The gas to be filled in the liposome may be a known gas without limitation. For example, carbon dioxide, helium, nitrogen, argon, sulfur hexafluoride or perfluorinated gas may be used. The gas is preferably a fluoride-containing fluoride, and examples thereof include perfluoropropane (C3F8), sulfur hexafluoride (SF6), perfluoropentane, decafluorobutane and perfluoro There is perfluorohexane.
상기 마이크로버블은 직경이 0.1~10㎛, 바람직하게는 1~10㎛일 수 있다.The micro bubble may have a diameter of 0.1 to 10 탆, preferably 1 to 10 탆.
상기 나노입자(20)는 내부에 약물(21)을 함유한다. 상기 나노입자는 알부민을 포함하여, 자기 집합체(self-aggregates)를 형성할 수 있다.The
상기 나노입자로는 응집성이 있는 단백질을 사용할 수 있으며, 바람직하게는 혈액 내에서 장기간 순환하면서도 응집 구조가 유지되어 약물을 안정적으로 전달할 수 있고, 암 표적성이 있는 공지된 알부민을 사용할 수 있다. 상기 알부민은 인간혈청 알부민 또는 이의 단편을 사용할 수 있다. 상기 나노입자는 직경이 156.02± 65.76nm, 바람직하게는 100~300nm일 수 있다.As the nanoparticles, a cohesive protein can be used. Preferably, the nanoparticles can circulate in the blood for a long period of time, maintain a cohesive structure, stably transfer the drug, and use known albumin having cancer targeting properties. The albumin may be human serum albumin or a fragment thereof. The nanoparticles may have a diameter of 156.02 ± 65.76 nm, preferably 100 to 300 nm.
상기 나노입자 내부에 함유되는 약물로는 이미 공지된 알부민에 적재될 수 있는 것들을 제한 없이 사용할 수 있다. 상기 약물로는 도세탁셀(Docetaxel), 시스플라틴(cis-platin), 캠토세신(camptothecin), 파클리탁셀(paclitaxel), 타목시펜(Tamoxifen), 아나스테로졸(Anasterozole), 글리벡(Gleevec), 5-플루오로우라실(5-FU), 플록슈리딘(Floxuridine), 류프로리드(Leuprolide), 플로타미드(Flutamide), 졸레드로네이트(Zoledronate), 독소루비신(Doxorubicin), 빈크리스틴(Vincristine), 젬시타빈(Gemcitabine), 스트렙토조토신(Streptozocin), 카보플라틴(Carboplatin), 토포테칸(Topotecan), 벨로테칸(Belotecan), 이리노테칸(Irinotecan), 비노렐빈(Vinorelbine), 히도록시우레아(hydroxyurea), 발루비신(Valrubicin), 레티노익산(retinoic acid) 계열, 메소트렉세이트(Methotrexate), 메클로레타민(Meclorethamine), 클로람부실(Chlorambucil), 부술판(Busulfan), 독시플루리딘(Doxifluridine), 빈블라스틴(Vinblastin), 마이토마이신(Mitomycin), 프레드니손(Prednisone), 테스토스테론(Testosterone), 미토산트론(Mitoxantron), 아스피린(aspirin), 살리실레이트(salicylates), 이부프로펜(ibuprofen), 나프로센(naproxen), 페노프로펜(fenoprofen), 인도메타신(indomethacin), 페닐부타존(phenyltazone), 시클로포스파미드(cyclophosphamide), 메클로에타민(mechlorethamine), 덱사메타손(dexamethasone), 프레드니솔론(prednisolone), 셀레콕시브(celecoxib), 발데콕시브(valdecoxib), 니메슐리드(nimesulide), 코르티손(cortisone) 및 코르티코스테로이드(corticosteroid)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. As the drug contained in the nanoparticles, those which can be loaded on the known albumin can be used without limitation. Such drugs include but are not limited to docetaxel, cis-platin, camptothecin, paclitaxel, tamoxifen, anasterozole, Gleevec, 5-fluorouracil (5-FU), Floxuridine, Leuprolide, Flutamide, Zoledronate, Doxorubicin, Vincristine, Gemcitabine ), Streptozocin, Carboplatin, Topotecan, Belotecan, Irinotecan, Vinorelbine, Hydroxyurea, Valrubicin, ), Retinoic acid series, methotrexate, Meclorethamine, Chlorambucil, Busulfan, Doxifluridine, Vinblastine Vinblastin, Mitomycin, Prednisone, Testostron (Testost) erogen, mitoxantron, aspirin, salicylates, ibuprofen, naproxen, fenoprofen, indomethacin, phenyl But are not limited to, phenyltazone, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide ), Cortisone, and corticosteroid. The term " corticosteroid "
상기 나노입자는 링커 또는 마이크로 버블 표면의 활성화된 반응기에 의해 마이크로버블에 결합될 수 있다. 상기 반응기로는 싸이올기(thiol), 아민기(amine)이거나, 링커로는 상기 반응기를 포함하는 화합물일 수 있다. The nanoparticles may be bound to microbubbles by an activated reactor of a linker or microbubble surface. The reactor may be a thiol or an amine or the linker may be a compound containing the above-mentioned reactor.
좀 더 구체적으로는 상기 마이크로버블과 상기 나노입자는 이들 사이에 아미드 결합(amide bond)될 수 있다. 상기 결합은 마이크로버블중의 카르복실기와 상기 알부민에 다수 포함된 아민기 사이에 일어나는 아미드 결합으로 형성될 수 있다. More specifically, the microbubble and the nanoparticles may be amide bonds between them. The bond may be formed by an amide bond occurring between a carboxyl group in the microbubble and an amine group contained in a majority of the albumin.
다른 양상에서, 본 발명은 마이크로버블과 약물을 함유하는 나노입자를 각각 제조하는 단계 ; 상기 나노입자와 상기 마이크로버블을 소정 비율로 물에 혼합하여 반응시키는 단계를 포함하는 초음파 조영제의 제조방법에 관계한다.In another aspect, the present invention provides a method of preparing nanoparticles comprising: preparing a microbubble and a drug-containing nanoparticle, respectively; Mixing the nanoparticles with the microbubbles in water at a predetermined ratio, and reacting the nanoparticles with water.
도 2는 본 발명의 초음파 조영제 제조 프로세스를 보여준다. 도 2를 참고하면, 상기 마이크로 버블을 제조하는 방법은 인지질을 유기용매와 혼합하여 지질박막을 형성하는 단계 ; 상기 지질박막을 물에 넣어 수화시키고, 여기에 가스를 주입 및 고압으로 유지하면서 초음파 처리하는 단계를 포함할 수 있다.2 shows a process for producing an ultrasound contrast agent of the present invention. Referring to FIG. 2, the method for preparing microbubbles comprises: mixing a phospholipid with an organic solvent to form a lipid thin film; And then subjecting the lipid thin film to water hydration, and then applying ultrasonic waves while injecting gas and maintaining the pressure at a high pressure.
바람직하게는, 상기 지질박막을 형성하는 단계는 인지질, NHS를 구비하는 인지질 유도체 및 유화제를 유기용매와 혼합하는 단계를 포함할 수 있다.Preferably, the step of forming the lipid thin film may include mixing a phospholipid, a phospholipid derivative having NHS, and an emulsifier with an organic solvent.
상기 지질박막을 형성하는 단계는 유화제 : 지질 : NHS를 구비하는 지질유도체의 몰비를 1~4 : 7~9 : 1~3 범위로 혼합할 수 있다.The step of forming the lipid thin film may include mixing the lipid derivative having the emulsifier: lipid: NHS in a molar ratio of 1: 4: 7 to 9: 1 to 3.
상기 인지질, NHS를 구비하는 인지질 유도체는 앞에서 상술한 양친매성 인지질 화합물을 사용할 수 있다.As the phospholipid and the phospholipid derivative having NHS, the amphiphilic phospholipid compound described above can be used.
상기 유화제로는 리소지질, 스테아르산, 폴리에틸렌글리콜, 폴리옥시에틸렌 지방산 에스테르, 폴리옥시에틸렌 지방산 스테아르레이트, 폴리옥시에틸렌 지방 알코올 등을 사용할 수 있다.Examples of the emulsifier include lysozyme, stearic acid, polyethylene glycol, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid stearate, and polyoxyethylene fatty alcohol.
상기 지질박막을 물에 넣어 수화시키고 가스를 충전하는 방법은 공지된 방법을 사용할 수 있으며, 예를 들면, 지질박막이 들어 있는 용기에 물, 글리콜, 글리세린 혼합액을 넣어 55~60℃의 온도를 유지하면서 녹이고, 여기에 가스를 200kPa로 넣어 초음파 처리할 수 있고, 또는 초음파와 mechanical agitation 방법을 병용하여 사용할 수 있다. For example, water, glycol, and glycerin mixed solution is added to a vessel containing a lipid thin film and maintained at a temperature of 55 to 60 ° C. , And the gas can be ultrasonicated by putting the gas at 200 kPa, or ultrasonic waves and mechanical agitation can be used in combination.
약물을 함유하는 나노입자를 제조하는 단계는 The step of preparing the drug-containing nanoparticles
알부민을 물에 녹인 후 여기에 약물을 주입하여 혼합물을 제조하는 단계, 상기 혼합물의 pH를 7~10, 바람직하게는 pH 8.0~8.5로 조절한 후 알코올류를 적하시키는 단계를 포함하고, 상기 단계에 의해 상기 알부민이 자기 집합체(self-aggregates)를 형성한다. Dissolving albumin in water to prepare a mixture by injecting a drug thereto, adjusting the pH of the mixture to 7 to 10, preferably to pH 8.0 to 8.5, and then dropping the alcohol, The albumin forms self-aggregates.
상기 나노입자와 상기 마이크로버블을 반응시키는 단계는 링커 또는 화학 반응기가 유도된 인지질을 이용하여 나노입자를 마이크로버블 표면에 결합시킬 수 있다. 상기 링커 또는 반응기로는 싸이올기(thiol), 아민기(amine) 바이오틴(biotin)-아비딘(avidin)등이 있을 수 있다.In the step of reacting the nanoparticles with the microbubble, the nanoparticles may be bonded to the microbubble surface using a phospholipid derived from a linker or a chemical reactor. The linker or the reactor may include a thiol group, an amine group, biotin-avidin group, or the like.
좀 더 구체적으로는 상기 마이크로버블과 상기 나노입자는 이들 사이에 아미드 결합(amide bond)될 수 있다. 상기 결합은 마이크로버블중의 카르복실기와 상기 알부민에 다수 포함된 아민기 사이에 일어나는 아미드 결합으로 형성될 수 있다. 좀 더 구체적으로는, 상기 마이크로버블 표면에 존재하는 NHS가 물에 가수분해되고, 마이크로버블 표면에 잔존하는 카르복실기와 상기 나노입자에 존재하는 아민기가 아미드 결합될 수 있다. More specifically, the microbubble and the nanoparticles may be amide bonds between them. The bond may be formed by an amide bond occurring between a carboxyl group in the microbubble and an amine group contained in a majority of the albumin. More specifically, the NHS present on the microbubble surface is hydrolyzed to water, and the carboxyl groups remaining on the microbubble surface and the amine groups present in the nanoparticles can be amide bonded.
상기 나노입자와 상기 마이크로버블의 혼합 비율은 결합 반응기의 몰 비로 1 : 0.5~2일 수 있다. The mixing ratio of the nanoparticles and the microbubbles may be 1: 0.5 to 2 in terms of the molar ratio of the coupling reactor.
이하, 본 발명의 이해를 돕기 위해 바람직한 실시예를 제시하지만, 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명이 이에 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following embodiments are provided for the purpose of easier understanding of the present invention, but the present invention is not limited thereto.
실시예Example 1 One
마이크로 Micro 버블bubble 제조 Produce
지질로는 1,2-disteraoyl-sn-glycero-3-phosphocholine(DSPC), DSPE-PEG2000-NHS(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]2000-N-hydroxysuccinimide), 유화제로는 Polyoxyethylene 40 stearate(POE40s)를 8:1:1의 molar ratio로 혼합하여 chloroform에 녹인 후, rotary evaporator를 이용하여 chloroform을 완전하게 증발시켜 지질박막을 형성하였다. Lipids include 1,2-disteraoyl-sn-glycero- 3-phosphocholine (DSPC), DSPE-PEG2000-NHS (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n- [poly (ethyleneglycol)] 2000 - N-hydroxysuccinimide) and
이어서, 증류수, 프로필렌글리콜, 글리세린을 8:1:1로 혼합한 후 이를 지질박막에 첨가하였다. 온도를 55-60℃를 유지하면서 지질을 녹였다. SF6 또는 C3F8 gas를 혼합액이 든 용기에 넣어 200kPa로 충진한 후 sonication 및 mechanical agitation을 통하여 마이크로버블을 제작하였다. Then, distilled water, propylene glycol, and glycerin were mixed at an ratio of 8: 1: 1 and then added to the lipid thin film. The lipids were dissolved while maintaining the temperature at 55-60 ° C. SF6 or C3F8 gas was filled in a container with a mixture of 200kPa and microbubbles were prepared by sonication and mechanical agitation.
나노입자 제조(Nanoparticle manufacturing ( HSAHSA -- NPsNPs ))
Human Serum Albumin(HSA)-40mg를 1mL 증류수에 녹인 후, Doxorubicin hydrochloride(5mg/mL)-100-200μL를 HSA가 녹아 있는 vial에 혼합하였다. KOH 또는 NaOH를 이용하여 혼합액의 pH를 8.0-8.5로 적정한 후, 에탄올 3-6mL을 1mL/min의 속도로 상기 혼합액에 적정하였다. HSA가 응집되어 혼합액이 혼탁해질 때 가교제인 8%-glutaraldehyde를 넣어 반응시켰다. 이어서, 에탄올을 완전히 증발시킨 후 남은 용액을 12000rpm, 4℃, 10min의 조건으로 원심분리시켰다. 가라앉은 pellet을 제외한 나머지 aliquot을 제거하였으며, 또한, 입자화되지 않은 HSA 및 Doxorubicin을 제거 한 후 증류수로 세척하였다. 3000rpm, 4℃, 5min의 조건으로 centrifugation하여 마이크로 크기의 입자를 제거하여 100-200nm 크기의 Doxorubicin이 적재된 HSA 나노입자(HSA-NPs)를 추출하였다.
Human Serum Albumin (HSA) -40 mg was dissolved in 1 mL of distilled water and 100-200 μL of Doxorubicin hydrochloride (5 mg / mL) was added to the vial containing HSA. The pH of the mixture was adjusted to 8.0-8.5 with KOH or NaOH, and 3-6 mL of ethanol was titrated into the mixture at a rate of 1 mL / min. When HSA coagulated and the mixture became turbid, 8% -glutaraldehyde, a cross-linking agent, was added and reacted. Subsequently, the ethanol was completely evaporated, and the remaining solution was centrifuged at 12,000 rpm, 4 ° C, and 10 min. The remaining aliquot was removed except for the settled pellet, and the non - granulated HSA and Doxorubicin were removed and washed with distilled water. (HSA-NPs) loaded with 100-200 nm Doxorubicin were extracted by centrifugation at 3000 rpm, 4 ° C, and 5 min.
마이크로버블과Microbubbles and 나노입자의 결합 Combination of nanoparticles
HSA 나노입자와 마이크로 버블을 1:0.5~2의 결합 반응기의 몰비로 상온에서 2시간 동안 혼합하여 아미드 결합으로 나노입자를 마이크로 버블에 결합하였다. 결합되지 않은 HSA 나노입자는 원심분리로 제거하였다. HSA nanoparticles and microbubbles were mixed at a molar ratio of 1: 0.5 ~ 2 at room temperature for 2 hours to bind the nanoparticles to microbubbles by amide bond. Unbound HSA nanoparticles were removed by centrifugation.
참고로, 마이크로 버블 제작 과정 중 손실된 NHS를 보충시켜주기 위하여 사전에 EDC와 NHS를 충분히 추가하여 반응시켜 원심분리기로 잔여 EDC, NHS를 세척하면 좀 더 결합효율을 높일 수 있다.
For reference, EDC and NHS are sufficiently added in advance to supplement the NHS lost during the microbubble production process, and the remaining EDC and NHS are washed with a centrifuge to increase the coupling efficiency.
비교예Comparative Example 1 One
실시예 1에서 제조한 마이크로버블을 비교예 1로 사용하였다(나노입자가 마이크로버블에 결합되지 않았음).
The microbubbles prepared in Example 1 were used as Comparative Example 1 (nanoparticles were not bound to microbubbles).
도 3은 본 발명의 마이크로 버블, 약물을 함유한 HSA 나노입자 및 이들이 결합된 초음파 조영제의 confocal fluorescence microscope 이미지이다. 도 4는 본 발명의 HSA 나노입자가 결합된 초음파 조영제의 크기 분포를 나타낸다.FIG. 3 is a confocal fluorescence microscope image of the microbubbles of the present invention, drug-containing HSA nanoparticles, and the ultrasound contrast agent bound thereto. FIG. 4 shows the size distribution of the ultrasound contrast agent to which the HSA nanoparticles of the present invention are bound.
도 3은 HSA-NPs의 결합을 fluorescence dye를 이용하여 결합 유무를 확인한 것으로, 형광염료(fluorescence dye)는 green color를 나타내는 FITC와 red color를 나타내는 DiIC18을 사용하였다. HSA-NPs입자 제조 시에 FITC가 결합된 bovine serum albumin을 10-20% 첨가하여 제작함으로써 HSA-NPs에 green color를 입혔으며 microbubble은 제조 시에 lipophilic한 DiIC18를 microbubble의 bilayer 사이에 적재함으로써 labeling 하였다. 도 3의 merged된 사진을 참고하면, HSA-NPs가 microbubble의 표면에 잘 붙어있음을 확인할 수 있다. FIG. 3 shows binding of HSA-NPs using fluorescence dye. FITC showing green color and DiIC18 showing red color were used for fluorescence dye. HSA-NPs were prepared by adding 10-20% FITC-conjugated bovine serum albumin to the HSA-NPs, and the microbubbles were labeled by loading lipophilic DiIC18 between microbubble bilayers . Referring to the merged picture of FIG. 3, it can be seen that the HSA-NPs stick to the surface of the microbubble.
도 4를 참고하면, HSA-NPs가 결합되지 않은 microbubble의 size distribution(gray)과 HSA-NPs가 결합된 microbubble의 size distribution(red)을 dynamic light scattering으로 측정한 것이다. HSA-NPs를 결합한 결과 사이즈 분포가 오른쪽으로 약간 shift되었음을 알 수 있으며, 사이즈가 1000~2000nm에 걸쳐 주로 분포되어 있음을 확인할 수 있다. Referring to FIG. 4, the size distribution (gray) of microbubbles not bound with HSA-NPs and the size distribution (red) of microbubbles combined with HSA-NPs were measured by dynamic light scattering. As a result of combining HSA-NPs, it can be seen that the size distribution is slightly shifted to the right, and it can be confirmed that the size is mainly distributed over 1000 to 2000 nm.
도 5는 실시예 1과 대조군인 water를 agarose 팬텀에 넣고 초음파 진단을 위한 상용화 기기를 이용하여 하모닉 모드로 초음파 영상을 확인한 결과이다. 초음파의 세기(mechanical index : MI)는 0.16으로 매우 약한 초음파를 사용하고, transducer는 5-12MHz의 초음파를 방출하는 것을 사용하였다. 도 5를 참고하면, water(거의 보이지 않음)에 비해 실시예 1(HSA-NPs가 결합된 microbubble)의 초음파 조영 효과가 매우 뛰어남을 알 수 있다. FIG. 5 is a result obtained by comparing ultrasound images in a harmonic mode using a commercial device for ultrasonic diagnosis by placing water, which is a control group, in Example 1 into an agarose phantom. The mechanical index (MI) of the ultrasonic wave was 0.16, and the ultrasonic wave of 5-12 MHz was used for the transducer. Referring to FIG. 5, it can be seen that the ultrasound imaging effect of Example 1 (HSA-NPs-bound microbubble) was excellent compared to water (almost invisible).
도 6은 초음파 조영제에 의하여 반사/투과된 초음파를 하이드로폰을 이용하여 초음파 성분을 수신, 이 때 수신된 성분 중 Harmonic 성분만을 검출하여 분석한 그래프이다. 본 실험에서는 단일 파장만을 방출할 수 있는 single transducer를 이용하였고, 주파수를 2MHz에서 5MHz까지 0.2MHz의 간격을 두고 동일한 세기(MI : 0.1, cycle :20)로 초음파를 방출하였다. Transducer에서 방출된 초음파는 agarose 팬텀에 담겨있는 제작된 HSA-NPs가 결합된 초음파 조영제에 의하여 반사/투과되며, 이러한 초음파를 하이드로폰을 이용하여 초음파 성분을 수신, 이 때 수신된 성분 중 Harmonic 성분만을 검출하여 분석하였다. 도 6을 참고하면, 3MHz에서 HSA-NPs가 결합된 microbubble이 가장 큰 harmonic 신호를 방출하였으며, 즉, 실시예 1의 HSA-NPs가 결합된 microbubble의 활성을 극대화 할 수 있는 초음파 주파수는 3MHz임을 도출해낼 수 있었다. 3MHz는 인체 내의 장기를 영상화하기에 문제없는 주파수이다. FIG. 6 is a graph of ultrasonic waves reflected / transmitted by an ultrasound contrast agent and receiving ultrasonic components using a hydrophone, wherein only the harmonic components of the received components are detected and analyzed. In this experiment, a single transducer capable of emitting only a single wavelength was used and ultrasonic waves were emitted at the same intensity (MI: 0.1, cycle: 20) at a frequency of 2 MHz to 5 MHz at intervals of 0.2 MHz. The ultrasound emitted from the transducer is reflected / transmitted by the ultrasonic contrast agent combined with the manufactured HSA-NPs contained in the agarose phantom. The ultrasonic wave is received by using the ultrasonic wave and the harmonic component And analyzed. Referring to FIG. 6, the microbubble coupled with HSA-NPs at 3 MHz emitted the greatest harmonic signal. That is, the ultrasound frequency capable of maximizing the activity of the HSA-NPs-bound microbubble of Example 1 was 3 MHz I could do it. 3MHz is a frequency that is no problem for imaging organs in the human body.
도 7은 3MHz의 초음파를 실시예 1의 HSA-NPs가 결합된 microbubble에 방출하여 수신한 피크(왼쪽)와 물에 방출하여 수신한 피크(오른쪽)과 비교한 그래프이다. 도 7을 참고하면, 두 번째 빨간색으로 표시된 peak가 harmonic 성분을 검출한 결과로 실시예 1의 초음파 조영제에서 수신한 피크가 물에서 수신한 피크보다 약 6dB 정도 높은데, 이것은 실시예 1이 물에 비해 초음파 수신 세기가 훨씬 높으므로 조영제로서 충분한 기능을 하고 있는 것으로 평가할 수 있다.FIG. 7 is a graph comparing an ultrasound of 3 MHz to a peak (left) emitted from a microbubble coupled with HSA-NPs of Example 1 and a peak received (right) emitted to water. Referring to FIG. 7, as a result of the detection of the harmonic component by the second red peak, the peak received by the ultrasound contrast agent of Example 1 is about 6 dB higher than the peak received by water, Since the ultrasound reception intensity is much higher, it can be estimated that it has sufficient function as a contrast agent.
도 8은 비교예 1(HSA-NPs가 결합되지 않은 microbubble)과 실시예 1에 대하여 장시간 초음파를 가했을 때의 초음파 조영제의 안정성을 측정한 결과를 나타낸다. 상기 안정성 측정은 도 5의 초음파 영상을 얻는 실험조건과 동일하게 진행하여 영상을 20 프레임으로 이미지를 촬영하여 영상의 intensity를 Matlab 프로그램을 이용하여 정량화하였다. 도 8을 참고하면, 시간이 경과할수록 Intensity가 감소하는데, 이것은 마이크로버블이 방출된 초음파에 의하여 파괴(burst)되기 때문이다. 실시예 1과 비교예 1은 Intensity의 감소가 거의 비슷한데, 즉, 실시예 1에 나노입자가 부착되었어도 마이크로버블의 안정성이나 영상효과가 저하되지 않음을 확인할 수 있다.
FIG. 8 shows the results of measuring the stability of an ultrasound contrast agent when Comparative Example 1 (microbubble without HSA-NPs) and Example 1 were subjected to long-term ultrasound. The stability measurement was performed in the same manner as in the experimental condition for obtaining the ultrasound image of FIG. 5, and the image was photographed at 20 frames, and the intensity of the image was quantified using the Matlab program. Referring to FIG. 8, the intensity decreases with time, because the microbubbles are burst by the emitted ultrasonic waves. In Example 1 and Comparative Example 1, the reduction of the intensities is almost the same. That is, even if the nanoparticles are attached in Example 1, it can be confirmed that the stability of the microbubbles and the image effect are not deteriorated.
세포수준에서의 항암효과 시험Anticancer effect test at cell level
항암제 paclitaxel을 함유하는 HSA-NPs를 이용하여 세포 수준에서의 항암효과를 검증하는 시험을 수행하였다. 사용된 세포는 MCF-7 세포로 6시간(도 9의 a), 24시간(도 9의 b), 72시간(도 9의 c)에 대한 항암효과를 검증하였으며, 특히 본 실험에서 도출된 최적화된 초음파 주파수인 3MHz의 초음파를 방출하여 세포내로의 침투효과 향상에 대한 검증실험도 함께 진행됨. 실험에서 진행된 군은 control, 비교예 1(HSA-NPs가 결합되지 않은 microbubble), paclitaxel을 함유하는 HSA-NPs(참고예), paxlitaxel을 함유하고 있는 HSA-NPs가 결합된 microbubble(실시예 1)로 총 4개의 군에 대하여 초음파를 조영한 군과 조영하지 않은 군으로 각각 나누어 진행하였다. 항암효과는 세포의 생존율을 검출할 때에 널리 이용되는 MTT assay로 분석하였으며, 그 실험결과를 도 9와 아래에 나타내었다. The HSA-NPs containing the anti-cancer drug paclitaxel were used to test the anti-cancer effect at the cellular level. The used cells were tested for anticancer activity against MCF-7 cells for 6 hours (FIG. 9 a), 24 hours (FIG. 9 b) and 72 hours (FIG. 9 c) The ultrasound frequency of 3 MHz, which is the ultrasound frequency, is emitted to verify the penetration effect into cells. Experimental groups were microbubbles (Example 1) in which the control, Comparative Example 1 (microbubble without HSA-NPs), HSA-NPs containing paclitaxel (Reference Example), HSA-NPs containing paxlitaxel We performed ultrasonography in four groups and non - contrast group, respectively. The anticancer effect was analyzed by MTT assay widely used for detecting cell survival rate, and the results of the experiment are shown in FIG. 9 and below.
(6h : control; 100.00± 2.41%, 111.76± 0.50% (US+), free-microbubble; 99.25± 1.82%, 94.38± 2.14% (US+), PTX loaded HSA-NPs; 75.90± 3.55%, 89.68± 3.64% (US+), PTX loaded HSA-NPs conjugated microbubble;87.35± 3.85%, 78.90± 5.93% (US+) (US +), PTX loaded HSA-NPs, 75.90 + - 3.55%, 89.68 + 3.64% (US +), PTX loaded HSA-NPs conjugated microbubble, 87.35 + 3.85%, 78.90 + 5.93% (US +),
24h control; 100± 7.28%, 80.01± 10.58% (US+), free-microbubble; 89.97± 10.68, 95.23± 9.59 (US+), PTX loaded HSA-NPs; 56.86± 2.26%, 52.39± 5.13% (US+), PTX loaded HSA-NPs conjugated microbubble; 69.72± 5.26%, 61.15± 6.54% (US+),24h control; 100 ± 7.28%, 80.01 ± 10.58% (US +), free-microbubble; 89.97 ± 10.68, 95.23 ± 9.59 (US +), PTX loaded HSA-NPs; 56.86 ± 2.26%, 52.39 ± 5.13% (US +), PTX loaded HSA-NPs conjugated microbubble; 69.72 ± 5.26%, 61.15 ± 6.54% (US +),
72h : control; 100.00± 0.08%, 94.67± 1.43% (US+), free-microbubble; 105.22± 2.72%, 85.28± 2.68% (US+), PTX loaded HSA-NPs; 26.37± 0.08%, 26.32± 1.25% (US+), PTX loaded HSA-NPs conjugated microbubble; 24.26± 0.05%, 16.53± 0.07% (US+)72h: control; 100.00 0.08%, 94.67 1.43% (US +), free-microbubble; 105.22 ± 2.72%, 85.28 ± 2.68% (US +), PTX loaded HSA-NPs; 26.37 ± 0.08%, 26.32 ± 1.25% (US +), PTX loaded HSA-NPs conjugated microbubble; 24.26 ± 0.05%, 16.53 ± 0.07% (US +)
도 9와 상기 실험결과를 참고하면, 약 72시간이 지난 후에는 암세포 생존율이 30%미만으로 떨어짐을 알 수 있으며, 즉 충분히 항암효과가 있음을 보여주고 있다. 다만, 참고예 1과 실시예 1은 비슷한 항암효과를 보여주고 있는데, 이것은 영상에 최적화된 3MHz의 주파수를 이용하였기 때문으로 보인다. 즉, 초음파 조영제에 의한 영상시간을 지속시키기 위해 낮은 세기(MI : 0.1)를 이용한 결과 초음파 조영제가 파괴되지 않아 세포벽에 pore를 형성시키지 않은 것으로 보인다. 참고로, 낮은 주파수의 초음파를 강하게 방출하여 실시예 1의 마이크로버블을 파괴한다면 세포벽에 기공이 형성(pore formation)되어 항암제가 세포벽 내부로 침투하는 효과가 증대되어 참고예보다 항암효과가 높을 것으로 예측할 수 있다.
Referring to FIG. 9 and the results of the experiment, it can be seen that after about 72 hours, the survival rate of the cancer cells is lowered to less than 30%, that is, it shows sufficient anticancer effect. However, Reference Example 1 and Example 1 show similar anticancer effects because of using an image-optimized 3 MHz frequency. In other words, low intensity (MI: 0.1) was used to maintain the imaging time by the ultrasound contrast agent, which did not destroy the ultrasound contrast agent and did not form pore on the cell wall. For reference, if the micro-bubble of Example 1 is destroyed by releasing a low-frequency ultrasonic wave strongly, pore formation in the cell wall (pore formation) increases the effect of the anticancer drug penetrating into the cell wall, .
동물실험Animal experiment
실시예 1과 비교예 1의 초음파 영상효과를 Balb/C nude mouse에 MCF-7을 이식한 질병 동물 모델을 이용하여 검증하였다. 각각의 물질을 intravenous injection하여 시간 별로 초음파 영상을 획득하였다. 이때 사용된 초음파 영상기기는 동물실험에 널리 이용되는 Visualsonics를 이용하였으며 사용된 주파수는 40MHz이다. injection하기 전의 초음파 영상을 기준으로 물질 주입시 초음파 조영제에 의해 영상 intensity가 증가되는 부분을 녹색 맵핑(green color mapping)하여 눈에 보기 쉽게 영상처리를 하여 도 10에 나타내었다. The ultrasound imaging effects of Example 1 and Comparative Example 1 were verified using Balb / C nude mice using an animal model of MCF-7 transplantation. Each material was injected intravenously to acquire ultrasound images every hour. The ultrasound imaging device used was Visualsonics, which is widely used in animal experiments, and the frequency used is 40 MHz. FIG. 10 shows a green color mapping of the portion where the intensity of the image is increased by the ultrasound contrast agent when the material is injected based on the ultrasound image before the injection, so that the image is easily visualized.
도 10을 참고하면, 실시예 1이 비교예 1보다 훨씬 뛰어난 영상효과를 보여주고 있음을 확인할 수 있다. 즉, 실시예 1에서는 시간이 경과하여도 영상효과가 지속되는 반면, 비교예 1은 곧바로 영상효과가 떨어짐을 알 수 있다.Referring to FIG. 10, it can be seen that Example 1 exhibits far superior image effects than Comparative Example 1. That is, in the first embodiment, the video effect continues even after the lapse of time, whereas the video effect of the first comparative example falls short.
도 11은 도 10의 영상처리 결과에 대하여 암에서의 intensity를 Matlab 프로그램을 이용하여 정량 비교한 그래프이다. 도 11에 의하면, 실시예 1이 비교예 1보다 암에서의 intensity가 훨씬 강하게 나타남을 확인할 수 있다. 11 is a graph showing a quantitative comparison of intensities in the arm using the Matlab program with respect to the image processing result of FIG. 11, it can be seen that the intensity of cancer in Example 1 is much stronger than that of Comparative Example 1.
도 12는 MCF-7 세포를 Balb/C nude mouse에 이식하여 동물 질병 모델을 제작하여 0.5mm의 크기를 보일 때 실험을 진행하여 mouse의 생존율을 관찰한 것이다. FIG. 12 shows the survival rate of the mouse when MCF-7 cells were transplanted into a Balb / C nude mouse and an animal disease model was prepared and the size of the animal was 0.5 mm.
각각의 그룹은 실험에서 진행된 군은 PBS를 주입한 군 (control), 비교예 1(HSA-NPs가 결합되지 않은 microbubble), paxlitaxel을 함유하고 있는 HSA-NPs가 결합된 microbubble(실시예 1)에 초음파를 가하지 않는 군(실시예 1-1), paxlitaxel을 함유하고 있는 HSA-NPs가 결합된 microbubble(실시예 1)에 초음파를 가한군(실시예 1-2)으로 총 4개의 그룹 (각 그룹당 N = 4)으로 진행을 하였으며 초음파를 가하는 방법은 초음파 영상실험에 사용된 기기인 Visualsonics를 이용하여 기기의 기능 중 하나인 microbubble destruction을 이용하여 마이크로 버블을 10분간 지속적으로 터뜨렸음. 본 실험은 3일 간격으로 반복하였으며 실험 동물은 매일 관찰하여 생존 여부를 확인하였다.In each group, the experimental group was treated with PBS (control), Comparative Example 1 (microbubble without HSA-NPs), microbubble with HSA-NPs containing paxlitaxel (Example 1) A total of 4 groups (each group) were divided into two groups: a group to which no ultrasonic waves were applied (Example 1-1), a group to which microspheres containing HSA-NPs containing paxlitaxel (Example 1) N = 4). Ultrasonography was performed using Visualsonics, a device used for ultrasound imaging, and microbubble destruction was continuously performed for 10 minutes using microbubble destruction. This experiment was repeated at intervals of 3 days. Animals were observed daily to confirm their survival.
실험 결과 control, 비교예 1은 약 10일 경과 후 한 마리씩 죽기 시작하였으며 23일 경과 대부분이 죽어 Control군과 비교예 1은 1마리, 만이 생존하였다. 실시예 1-1은 15일째 한 마리가 사멸하였으며 23일 후 또 한 마리가 사멸하였으며, 실시예 1-2는 21일에 한 마리가 사멸할 뿐 실험이 진행 동안 3마리가 생존하였다. As a result of control, in Comparative Example 1, one dog died after about 10 days. Most of the mice died after 23 days, and only one control group and Comparative Example 1 survived. In Example 1-1, one dog died on the 15th day and another dog died after 23 days. In Example 1-2, one dog died on the 21st day, and three animals survived during the experiment.
실시예 1-1은 1-2에 비해 항암효과가 다소 떨어지는데, 이것은 큰 마이크로 버블에 붙어 있는 PTX(paxlitaxel)-HSA-NP는 혈관 조직을 통하여 암 조직으로 Human serum albumin 나노입자가 침투해 들어가지 못하고 혈관에서 항암제를 방출함으로써 암세포내로 전달 효능이 떨어지기 때문이다.PTX (paxlitaxel) -HSA-NP attached to a large microbubble penetrates into the cancer tissue through the vascular tissue and permeates into human serum albumin nanoparticles. And the release of anticancer drugs from the blood vessels deteriorates the delivery efficiency into cancer cells.
반면, 실시예 1-2(PTX-HSA-NPs-MB(US+))의 경우에는 암 조직 주변에서 초음파를 이용하여 마이크로 버블을 터뜨림으로써 나노 크기의 human serum albumin 나노입자가 혈관 조직을 효과적으로 통과하여 암 조직으로 전달되어 효과적인 항암 효과를 보인 것으로 판단된다. 또한 human serum albumin 나노입자는 gp60 리셉터(receptor)에 의해서 transcytosis 됨으로써 암세포 내로 효과적으로 전달되어 항암 효과를 향상시킬 수 있었다. 또한 마이크로 버블이 외부에서 전달된 초음파에 의해 공동현상(cavitation)이 발생됨으로서 암 조직 세포벽에 기공을 형성하여 전달 효과를 상승시킨 것으로 판단된다.On the other hand, in the case of Example 1-2 (PTX-HSA-NPs-MB (US +)), microbubbles were fired using ultrasonic waves around cancer tissues, so that nanosized human serum albumin nanoparticles effectively passed through blood vessel tissues And it was transferred to cancer tissues, thus showing an effective anti-cancer effect. In addition, human serum albumin nanoparticles were transcytosed by the gp60 receptor and were effectively transferred into cancer cells, thereby improving the anti-cancer effect. In addition, cavitation is generated by ultrasonic waves transmitted from the outside of microbubbles, and it is considered that pores are formed in the cancer tissue cell wall and the transfer effect is increased.
본 발명의 단순한 변형 내지 변경은 모두 본 발명의 영역에 속하는 것으로, 본 발명의 구체적인 보호범위는 첨부된 특허청구범위에 의하여 명확해질 것이다.
It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.
10 : 마이크로버블 20 : 나노입자
30 : 링커 21 : 약물 10: micro bubble 20: nanoparticle
30: Linker 21: Drug
Claims (15)
상기 마이크로 버블의 표면에 결합된 약물을 함유한 나노입자를 포함하고, 상기 나노입자는 링커 또는 마이크로 버블 표면의 활성화된 반응기에 의해 상기 마이크로버블에 결합되고, 여기서 상기 반응기는 싸이올기(thiol) 또는 카르복실기이고, 상기 링커는 상기 반응기를 포함하는 화합물이고, 또는
상기 마이크로버블과 상기 나노입자의 결합은 아미드 결합(amide bond), 싸이올기 (thiol)간의 결합 또는 바이오틴(biotin)-아비딘(avidin) 결합이고,
상기 마이크로버블은 인지질 기반의 리포좀 코어(core) 부분이 가스로 충진된 것을 특징으로 하는 초음파 조영제. A micro bubble having a gas filled therein; And
Wherein the nanoparticles are bound to the microbubble by an activated reactor of a linker or a microbubble surface, wherein the reactor is a thiol or The linker is a compound containing the above-mentioned reactor, or
The bond between the microbubble and the nanoparticle is an amide bond, a bond between a thiol or a biotin-avidin bond,
Wherein the microbubble comprises a phospholipid-based liposome core portion filled with a gas.
상기 나노입자와 상기 마이크로버블을 소정 비율로 물에 혼합하여 반응시키는 단계를 포함하고,
상기 마이크로버블을 제조하는 단계는 인지질, 인지질 유도체 및 유화제를 유기용매와 혼합하여 지질박막을 형성하는 단계 및 상기 지질박막을 물에 넣어 수화시키고, 여기에 가스를 주입하는 단계를 포함하고,
상기 반응 단계는 상기 나노입자가 링커 또는 마이크로 버블 표면의 활성화된 반응기에 의해 상기 마이크로버블에 결합되고, 여기서, 상기 반응기는 싸이올기(thiol) 또는 카르복실기이고, 상기 링커는 상기 반응기를 포함하는 화합물일이고, 또는
상기 반응 단계는 아미드 결합(amide bond), 바이오틴(biotin)-아비딘(avidin) 결합이나 싸이올기(thiol) 간의 결합에 의해 상기 마이크로버블에 상기 나노입자가 결합되는 단계인 것을 특징으로 하는 초음파 조영제의 제조방법.Preparing microbubbles and nanoparticles containing the drug, respectively;
Mixing the nanoparticles and the microbubbles in water at a predetermined ratio and reacting them,
The step of preparing microbubbles comprises the steps of forming a lipid thin film by mixing a phospholipid, a phospholipid derivative and an emulsifier with an organic solvent, and hydrating the lipid thin film into water and injecting gas thereto,
The reaction step Wherein said nanoparticles are bound to said microbubble by an activated reactor of a linker or microbubble surface, wherein said reactor is a thiol or carboxyl group, said linker being a compound comprising said reactor, or
Wherein the reaction step is a step of binding the nanoparticles to the microbubble by binding between an amide bond, a biotin-avidin bond or a thiol. Gt;
알부민을 물에 녹인 후 여기에 약물을 주입하여 혼합물을 제조하는 단계 ;
상기 혼합물의 pH를 7~9로 조절한 후 알코올류를 적하시키는 단계로서, 상기 단계에 의해 상기 알부민이 자기 집합체(self-aggregates)를 형성하는 것을 특징으로 하는 초음파 조영제의 제조방법.11. The method of claim 10, wherein the step of preparing the drug-containing nanoparticles comprises
Dissolving albumin in water and injecting a drug thereto to prepare a mixture;
Adjusting the pH of the mixture to 7 to 9, and then dropping the alcohol, wherein the albumin forms self-aggregates.
11. The method of claim 10, wherein the mixing ratio of the nanoparticles to the microbubbles is 1: 0.5 to 2 in a molar ratio of the coupling reactor.
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