KR101080499B1 - Hollow Nanoparticles Containing Paramagnetic Material as MRI Contrast Agent and Drug Delivery System Using The Same - Google Patents
Hollow Nanoparticles Containing Paramagnetic Material as MRI Contrast Agent and Drug Delivery System Using The Same Download PDFInfo
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- KR101080499B1 KR101080499B1 KR1020090030481A KR20090030481A KR101080499B1 KR 101080499 B1 KR101080499 B1 KR 101080499B1 KR 1020090030481 A KR1020090030481 A KR 1020090030481A KR 20090030481 A KR20090030481 A KR 20090030481A KR 101080499 B1 KR101080499 B1 KR 101080499B1
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Abstract
본 발명은 상자성 물질을 포함하는 중공형 나노입자로 이루어진 MRI(자기공명영상) 조영제 및 이의 제조방법을 제공한다. 상기 MRI 조영제는 약물 봉입 용량이 커 약물 전달 시스템 및 약물 전달 캐리어로 사용될 수 있고, 이완성이 우수하여 T 1의 효과가 강조된 MRI 및 T 2의 효과가 강조된 MRI 모두에 이용될 수 있다. The present invention provides an MRI (magnetic resonance imaging) contrast agent consisting of hollow nanoparticles containing paramagnetic material and a method of manufacturing the same. The MRI contrast agent may be used as a drug delivery system and a drug delivery carrier because of a large drug loading capacity, and may be used in both MRIs in which the effect of T 1 is emphasized and MRI in which the effect of T 2 is emphasized.
조영제, 약물전달, 자기공명, 망간, 중공형, 나노입자 Contrast Agents, Drug Delivery, Magnetic Resonance, Manganese, Hollow, Nanoparticles
Description
본 발명은 상자성 물질을 포함하는 중공형 나노입자로 이루어진 MRI 조영제, 이의 제조방법 및 이를 이용한 약물 전달 캐리어 및 약물 전달 시스템에 관한 것이다. The present invention relates to an MRI contrast agent consisting of hollow nanoparticles containing paramagnetic material, a preparation method thereof, and a drug delivery carrier and a drug delivery system using the same.
나노 크기의 콜로이드 입자는 작은 크기와 큰 표면적을 가지기 때문에 자기공명영상(Magnetic Resonance Imaging; MRI)의 조영제로 사용될 때 여러 가지 장점이 있다. 예를 들면, 활성 자기 중심부의 큰 페이로드(payload)를 운반하는 능력이 있고 생체막 시스템에 쉽게 침투할 수 있으며, 혈액에서의 순환 시간이 길고 친화성이 있는 분자에 효과적으로 결합하고, 그리하여 저농도에서도 특정 타겟에 대한 섬세한 영상을 제공할 수 있게 해 주는 등의 장점이 있다(J. W. M. Bulte et al., NMR Biomed .2004, 17, 484; 및 S. Mornet S. et al., J. Mater. Chem . 2004, 14, 2161). Nano-sized colloidal particles have a number of advantages when used as contrast medium for magnetic resonance imaging (MRI) because of their small size and large surface area. For example, it has the ability to carry large payloads in the active magnetic core and can easily penetrate biological membrane systems, and efficiently binds to molecules with long affinity and long affinity in the blood, so that even at low concentrations Etc. (JWM Bulte et al., NMR Biomed . 2004, 17, 484; and S. Mornet S. et al. , J. Mater. Chem . 2004) . , 14, 2161).
또한, 나노입자는 치료제와 결합하여 2가지 기능, 즉, MRI 조영의 역할 및 약물전달을 동시에 할 수 있는 의학 시스템으로서 이용될 수 있다(J. Kim et al., Adv . Mater. 2008, 20, 478). 예를 들면, 초상자성 산화철 나노입자는 효과가 좋은 T 2 조영제로 개발되어서 종양, 줄기세포 이동 및 암 전이 영상을 찍는 데 이용되고 있다(M. G. Harisinghani et al., J. Med . 2003, 348, 2491. ). 최근 일부 가돌리듐 이온(Gd3 +) 또는 망간 이온(Mn2 +)을 포함하는 콜로이드 나노입자는 잠재적인 T 1 MRI 조영제로 평가받고 있다(S. Flacke et al., Circulation 2001, 104, 1280). In addition, nanoparticles can be used as a medical system that can be combined with a therapeutic agent to simultaneously perform two functions, namely the role of MRI contrast and drug delivery (J. Kim et al., Adv . Mater. 2008, 20, 478). For example, superparamagnetic iron oxide nanoparticles have been developed as effective T 2 contrast agents and used for imaging tumor, stem cell migration and cancer metastasis (MG Harisinghani et al., J. Med . 2003, 348, 2491 .). Recently some gadol colloidal nanoparticle containing lithium ions (Gd + 3) or manganese ions (Mn + 2) have been evaluated as potential MRI contrast agents T 1 (S. Flacke et al., Circulation 2001, 104, 1280) .
최근, 마그네틱 이온을 포함하는 중공형 나노입자가 다양한 제조방법을 통해서 제조되고 있지만, 이들을 의학적으로 적용한 경우는 거의 없었다(J. H. Gao et al., Chem. Soc .2006, 128, 12632; Y. Yin, R. N. Rioux, C. K. Erdonmez, S. Hughes, G. A. Somorjai, A. P. Alivisatos, Science 2004, 304, 711.). 또한, 가돌리듐 이온(Gd3 +) 또는 망간 이온(Mn2 +)과 같이 잠재적으로 독성을 띠는 물질의 농도가 매우 낮은 상태에서도 작동할 수 있는, 이완성이 매우 강화된 MRI 조영제용 나노 입자에 대한 요구가 증가하고 있다. Recently, hollow nanoparticles containing magnetic ions have been produced through various manufacturing methods, but few of them have been applied medically (JH Gao et al., Chem. Soc . 2006, 128, 12632; Y. Yin, RN Rioux, CK Erdonmez, S. Hughes, GA Somorjai, AP Alivisatos, Science 2004, 304, 711.). In addition, the gadol lithium ions (Gd 3 +) or manganese ions (Mn 2 +) nanoparticles for MRI contrast agents potentially capable to work in the strip is very low concentrations of substances state toxic, flaccid highly strengthened as The demand for it is increasing.
이에 따라, 본 발명자들은 중공형 나노 입자로서, 이완성이 우수한 MRI 조영제를 개발하였고, 이를 이용하여 대용량의 약물 봉입이 가능한 약물 전달 시스템을 완성하였다. Accordingly, the present inventors developed an MRI contrast medium having excellent relaxation properties as hollow nanoparticles, and completed a drug delivery system capable of encapsulating a large amount of drugs using the same.
본 발명은 T 1의 효과가 강조된 MRI(자기공명영상) 및 T 2의 효과가 강조된 MRI 모두에 사용될 수 있는, MRI 이완성 및 약물 봉입 용량이 모두 우수한 MRI 조영제, 이의 제조 방법 및 상기 조영제 를 이용한 약물 전달 시스템 및 약물 전달 캐리어를 제공하기 위한 것이다. The present invention can be used for both MRI (Magnetic Resonance Imaging) with the effect of T 1 and MRI with the effect of T 2 , which has excellent MRI relaxation and drug loading capacity, a preparation method thereof, and a drug using the contrast agent. To provide a delivery system and a drug delivery carrier.
본 발명은 상자성 물질을 포함하는 중공형 나노입자로 이루어진 자기공명영상(MRI) 조영제에 관한 것이다. The present invention relates to a magnetic resonance imaging (MRI) contrast agent consisting of hollow nanoparticles containing paramagnetic material.
본 발명의 다른 양태는, 상자성 물질을 포함하는 중공형 나노입자로 이루어져 있고, 상기 나노 입자가 생체적합성 물질로 피복되어 있는 것을 특징으로 하는, 자기공명영상(MRI) 조영제다. Another aspect of the present invention is a magnetic resonance imaging (MRI) contrast medium comprising hollow nanoparticles containing paramagnetic material, wherein the nanoparticles are coated with a biocompatible material.
상기 조영제에 있어서, 상기 생체적합성 물질이 폴리비닐알콜, 폴리락타이드(polylactide), 폴리글리콜라이드(polyglycolide), 폴리락타이드글리콜라이드공중합체(poly(lactide-co-glycolide)), 폴리안하이드라이드(polyanhydride), 폴리에스테르(polyester), 폴리에테르에스테르(polyetherester), 폴리카프로락톤(polycaprolactone), 폴리에스테르아마이드(polyesteramide), 폴리아크릴레이트(polyacrylate), 폴리우레탄(polyurethane), 폴리비닐플루오라이드(polyvinyl fluoride), 폴리비닐이미다졸(poly(vinyl imidazole)), 클로로술포네이트 폴리올레 핀(chlorosulphonate polyolefin), 폴리에틸렌옥사이드(polyethylene oxide), 폴리에틸렌글리콜(poly(ethylene glycol)) 및 덱스트란(dextran)으로 이루어진 군에서 선택되는 어느 하나, 이들의 혼합물 또는 이들의 공중합체를 포함하는 것이 바람직하다. 또한, 상기 생체적합성 물질이 폴리에틸렌글리콜인 것이 보다 바람직하다. In the contrast agent, the biocompatible material is polyvinyl alcohol, polylactide, polyglycolide, polylactide glycolide copolymer (poly (lactide-co-glycolide)), polyanhydride (polyanhydride), polyester, polyetherester, polycaprolactone, polyesteramide, polyacrylate, polyurethane, polyvinyl fluoride fluoride, poly (vinyl imidazole), chlorosulphonate polyolefin, polyethylene oxide, polyethylene glycol and dextran It is preferred to include any one selected from the group consisting of, mixtures thereof or copolymers thereof. In addition, the biocompatible material is more preferably polyethylene glycol.
본 발명의 또 다른 양태는, 상자성 물질을 포함하는 중공형 나노입자에 약물이 봉입되어 있는 것을 특징으로 하는, 세포 내 약물 전달 캐리어(carrier)용 나노입자이다. Another aspect of the invention is a nanoparticle for intracellular drug delivery carrier, characterized in that the drug is enclosed in a hollow nanoparticle containing a paramagnetic material.
본 발명의 또 다른 양태는, 상자성 물질을 포함하는 중공형 나노입자로 이루어진 MRI 조영제 상기 나노입자에 봉입되는 약물 및 상기 약물이 봉입된 나노입자로 이루어진 MRI 조영제가 투여되는 생체 조직을 포함하여 이루어지는, 세포 내 약물 전달 시스템이다. Another embodiment of the present invention, MRI contrast agent consisting of hollow nanoparticles containing paramagnetic material comprising a biological tissue to which the drug encapsulated in the nanoparticles and the MRI contrast agent consisting of the nanoparticles enclosed with the drug is administered, Intracellular drug delivery system.
상기 약물 전달 시스템에서, 상자성 물질은 산화 망간(IV)인 것이 바람직하다. In the drug delivery system, the paramagnetic material is preferably manganese oxide (IV).
상기 나노입자 및 약물전달 시스템에서, 나노입자에 봉입되는 약물은 DOX(doxorubicin)인 것이 바람직하다. In the nanoparticle and drug delivery system, the drug encapsulated in the nanoparticle is preferably DOX (doxorubicin).
본 발명의 또 다른 양태는 상기 중공형 나노입자로 이루어진 자기공명영상(MRI) 조영제를 사용하는 것을 특징으로 하는, 자기공명영상(MRI) 조영 방법이다. Another aspect of the present invention is a magnetic resonance imaging (MRI) imaging method, characterized in that using the magnetic resonance imaging (MRI) contrast agent consisting of the hollow nanoparticles.
본 발명의 또 다른 양태는,Another aspect of the invention,
(a) 상자성을 가지는 균일한 금속 산화물 나노입자를 제조하는 단계;(a) preparing uniform metal oxide nanoparticles having paramagnetic;
(b) 상기 균일한 금속 산화물 나노입자를 증류수에 분산시켜서 저장하는 단계;(b) dispersing and storing the uniform metal oxide nanoparticles in distilled water;
(c) 상기 증류수에 분산된 균일한 금속 산화물 나노입자를 산성 용액에 넣고 교반하는 단계; 및(c) putting uniform metal oxide nanoparticles dispersed in the distilled water into an acidic solution and stirring; And
(d) 상기 교반된 용액을 원심분리하여, 상자성 물질을 포함하는 중공형 나노입자를 수득하는 단계를 포함하는 것을 특징으로 하는, 중공형 나노입자로 이루어진 MRI 조영제의 제조방법이다. (d) centrifuging the stirred solution to obtain hollow nanoparticles comprising paramagnetic material, wherein the MRI contrast agent comprising hollow nanoparticles is prepared.
상기 방법에서, 균일한 금속 산화물 나노입자가 균일한 산화망간 나노입자인 것이 바람직하다. 또한, 단계 (a)에서 망간-올레이트의 열분해 단계 및 폴리에틸렌글라이콜 포스포리피드를 이용한 캡슐화 과정을 통해 나노 입자를 제조하고, 단계 (b)에서 상기 균일한 산화망간 나노입자를 증류수에 분산시켜서 1 내지 40일 동안 저장하고, 단계 (c)에서 상기 증류수에 분산된 균일한 산화망간 나노입자를 프탈레이트 완충액에 넣고 교반하여, 상기 나노입자의 껍질에 해당하는 산화망간(Mn3O4)은 남겨두고 내부의 산화망간(MnO)을 제거하는 것이 바람직하다. In the above method, it is preferable that the uniform metal oxide nanoparticles are uniform manganese oxide nanoparticles. In addition, nanoparticles are prepared by the thermal decomposition step of manganese-oleate and encapsulation using polyethyleneglycol phosphide in step (a), and the uniform manganese oxide nanoparticles are dispersed in distilled water in step (b). And store for 1 to 40 days, and in step (c), the manganese oxide nanoparticles dispersed in the distilled water are put in a phthalate buffer and stirred, and the manganese oxide (Mn 3 O 4 ) corresponding to the shell of the nanoparticles is It is desirable to remove the manganese oxide (MnO) inside.
본 발명의 중공형 나노입자에서, 상기 상자성 물질은 망간, 가돌리늄, 에르븀, 크롬, 철, 코발트, 니켈, 란탄계열 원소, 악티늄계열 원소로 이루어진 군으로부터 선택되는 것이 바람직하다. 또한, 상기 상자성 물질은 망간인 것이 보다 바람직하다. In the hollow nanoparticles of the present invention, the paramagnetic material is preferably selected from the group consisting of manganese, gadolinium, erbium, chromium, iron, cobalt, nickel, lanthanum-based elements, and actinium-based elements. In addition, the paramagnetic material is more preferably manganese.
본 발명의 중공형 나노입자에서, 상기 상자성 물질은 망간, 가돌리늄, 에르 븀, 크롬, 철, 코발트, 니켈, 란탄계열 원소, 악티늄계열 원소로 이루어진 군으로부터 선택되는 것이 바람직하다. 또한, 상기 상자성 물질은 망간 이온인 것이 보다 바람직하다. In the hollow nanoparticles of the present invention, the paramagnetic material is preferably selected from the group consisting of manganese, gadolinium, erbium, chromium, iron, cobalt, nickel, lanthanum-based elements, and actinium-based elements. In addition, the paramagnetic material is more preferably manganese ions.
본 발명의 중공형 나노입자에서, 상기 상자성 물질은 망간, 가돌리늄, 에르븀, 크롬, 철, 코발트, 니켈, 란탄계열 원소, 악티늄계열 원소로 이루어진 군으로부터 선택되는 것이 바람직하다. 또한, 상기 상자성 물질은 망간 이온을 포함하는 킬레이트 화합물인 것이 보다 바람직하다. In the hollow nanoparticles of the present invention, the paramagnetic material is preferably selected from the group consisting of manganese, gadolinium, erbium, chromium, iron, cobalt, nickel, lanthanum-based elements, and actinium-based elements. In addition, the paramagnetic material is more preferably a chelate compound containing manganese ions.
본 발명의 중공형 나노입자에서, 나노입자의 내부 직경이 2 내지 20 nm이고, 외부 직경이 5 내지 30 nm인 것이 바람직하다. In the hollow nanoparticles of the present invention, it is preferable that the inner diameter of the nanoparticles is 2 to 20 nm and the outer diameter is 5 to 30 nm.
본 발명의 중공형 나노입자는 상자성 물질을 포함하는 껍질(shell) 및 상기 껍질 내부의 속이 비어 있는 코어(core)로 이루어진 속이 빈(hollow) 형태를 갖는다. 이때, 껍질의 직경을 나노입자의 외부 직경, 코어의 직경을 나노입자의 내부 직경이라고 한다. Hollow nanoparticles of the present invention It has a hollow form consisting of a shell containing paramagnetic material and a hollow core inside the shell. At this time, the diameter of the shell is referred to as the outer diameter of the nanoparticles, the diameter of the core is referred to as the inner diameter of the nanoparticles.
본 발명의 상자성 물질을 포함하는 중공형 나노입자로 이루어진 MRI 조영제는 MRI 라벨링, 이완성이 우수하고 약물 봉입 용량이 클 뿐만이 아니라 제조방법이 간단하다. The MRI contrast agent consisting of hollow nanoparticles containing paramagnetic material of the present invention is excellent in MRI labeling, relaxation, large drug encapsulation capacity, and simple manufacturing method.
구체적으로, 본 발명에 따른 MRI 조영제 는 중공형으로, 코어(core)가 비어있으므로, 빈 공간으로 고분자량의 약물들이 들어갈 수 있어 약물 봉입용량(drug loading capacity)이 클 뿐만 아니라, 세포 섭취 능력(cellular uptake)이 우수하 다. 또한, 물과 접촉할 수 있는 표면적(water-accessible surface area)이 넓다. 따라서, MR-활성 자기 중심부에 큰 페이로드를 운반할 수 있어, 항암제 등의 약물을 전달하기 위한, 약물 전달 시스템 또는 약물 전달 캐리어로 이용될 수 있다. 또한, 본 발명에 따른 MRI 조영제 는 이완성이 우수하며, T 1의 효과가 강조된 MRI 및 T 2의 효과가 강조된MRI 모두에 사용될 수 있는 듀얼(dual) 조영제 로 이용될 수 있다. 이에 따라, 본 발명에 따른 나노입자로 이루어진 조영제 는 치료를 위한 진단 영상 및 약물 전달을 결합한 치료/진단(theragnosis)용의 이중기능을 가지는 MRI 약제 개발에 대한 새로운 접근법을 제공할 수 있다. Specifically, since the MRI contrast agent according to the present invention is hollow, the core is empty, high molecular weight drugs can enter the empty space, and thus the drug loading capacity is large, and the cell intake capacity ( cellular uptake is excellent. In addition, the water-accessible surface area in contact with water is large. Therefore, it is possible to carry a large payload in the MR-active magnetic core, which can be used as a drug delivery system or drug delivery carrier for delivering drugs such as anticancer drugs. In addition, the MRI contrast agent according to the present invention is excellent in relaxation, it can be used as a dual contrast medium that can be used in both MRI with the effect of T 1 and MRI with the effect of T 2 . Accordingly, the contrast agent consisting of nanoparticles according to the present invention may provide a new approach to the development of a dual function MRI drug for treatment / theragnosis combined with diagnostic imaging and drug delivery for treatment.
이하 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 하기 실시예는 본 발명의 예시 목적을 위한 것이며, 첨부된 특허청구범위에 따른 본 발명의 보호범위를 제한하고자 하는 것은 아니다. Through the following examples will be described the present invention in more detail. The following examples are for illustrative purposes of the invention and are not intended to limit the protection scope of the invention in accordance with the appended claims.
실시예Example
실시예Example 1. 중공형 산화망간 나노입자( 1. Hollow manganese oxide nanoparticles HMONHMON ) 제조 및 그 특성A) manufacture and its characteristics
1.1. 일반적인 고려 사항1.1. General Considerations
MnCl2 ·H2O(Aldrich), 올레산염 나트륨(TCI), 올레산(불포화 지방산; Aldrich), 프탈레이트 완충액(pH 4.6, (주)삼전화학) 및 1,2-디스테아로일-sn-글라이세로-3-포스포에탄올아민-N-[메톡시(폴리에틸렌 글라이콜)-2000] (mPEG-2000 PE, Avanti Polar Lipids, Inc.)을 포함한 반응물을 구매한 후 정제없이 사용하였다. 투과전자현미경(TEM)으로 JEOL JEM-2010를 사용하였고, 주사터널현미경(SEM, scanning tunneling microscopy)으로 LEO SUPRA 55(Carl Zeiss, Germany)을 사용하였다. 나노입자의 자기적 성질을 5 T의 초전도자석을 갖추고 있는 초전도양자간섭소자(SQUID, superconducting quantum interference device) 자력계(Quantum Design, MPMS5XL)에 의해 측정하였다. X-ray 광전자 분광기로 K-알파(Thermo Electron, U.K.)를 이용하였다. MnCl 2 · H 2 O (Aldrich ), sodium oleate (TCI), oleic acid (unsaturated fatty acid; Aldrich), phthalate buffer solution (pH 4.6, (Note) Samchun Chemical) and 1,2-discharge days Te -sn- article Reactions including Lysero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (mPEG-2000 PE, Avanti Polar Lipids, Inc.) were purchased and used without purification. JEOL JEM-2010 was used as a transmission electron microscope (TEM) and LEO SUPRA 55 (Carl Zeiss, Germany) was used as a scanning tunneling microscopy (SEM). The magnetic properties of the nanoparticles were measured by a superconducting quantum interference device (SQUID) magnetometer (Quantum Design, MPMS5XL) equipped with a superconducting magnet of 5 T. K-alpha (Thermo Electron, UK) was used as an X-ray photoelectron spectrometer.
1.2.1.2. HMONHMON 합성 synthesis
올레인산으로 안정화된 직경 약 20 nm의 산화망간 나노입자(Manganese oxide nanoparticles; MON) 및 균일한 산화망간 나노입자(water-dispersible manganese; WMON)를, 망간-올레이트 복합체의 열분해 및 폴리에틸렌글라이콜 포스포리피드(polyethyleneglycol phospholipid)를 이용한 캡슐화(emcapsulation)를 통해 제조하였다(Na, H. B. et al., Angew. Chem. Int. Ed. 2007, 46, 5397; Park, J. et al., Nat. Mater. 2004, 3, 891.; Dubertret, B. et al., Science 2002, 298, 1759. 등 참고 ). 상기 과정을 도식화하여 도 2에 나타내었다. Manganese oxide nanoparticles (MON) and uniform water-dispersible manganese (WMON) of about 20 nm in diameter stabilized with oleic acid were subjected to thermal decomposition and polyethylene glycol force of manganese-oleate complexes. It was prepared by encapsulation using polyglycol phospholipid (Na, HB et al., Angew. Chem. Int. Ed. 2007, 46, 5397; Park, J. et al., Nat. Mater. 2004, 3, 891; see Dubertret, B. et al., Science 2002, 298, 1759. et al . The process is schematic and is shown in FIG. 2.
에탄올, 물 및 n-헥산의 혼합 용매 내에서 MnCl2 ·H2O와 올레산염 나트륨을 반응시켜 제조된 1.24 g의 Mn-올레산염 복합체(2 mmol)를 10 g의 1-옥타데센 (Aldrich Chemical Co., 90%)에 용해시켰다. 상기 혼합 용액을 진공 상태에서 70 ℃, 1 시간 동안 가스를 제거하여 물과 산소를 제거하고 강하게 교반하면서 300 ℃까지 가열하고, 1 시간 동안 온도를 유지한 후 실온으로 냉각시켰다. 20 ml의 헥산이 나노입자의 분산성을 향상시키기 위해 첨가된 후, 나노입자들을 침전시키기 위해 80 ml의 아세톤을 첨가하였다. 상기 왁스형 침전물을 원심분리에 의해 회수하였다. 상기 정제 과정을 과잉 계면활성제와 용매를 제거하기 위해 2 회 이상 반복하였다. Ethanol, 1-octadecene in 10 g water and n- hexane 2 · H 2 O and the MnCl oleate with 1.24 g of Mn- oleate complex (2 mmol) prepared by the reaction of sodium in a mixture of (Aldrich Chemical Co., 90%). The mixed solution was degassed at 70 ° C. in a vacuum for 1 hour to remove water and oxygen, heated to 300 ° C. with vigorous stirring, maintained at temperature for 1 hour and then cooled to room temperature. After 20 ml of hexane was added to improve the dispersibility of the nanoparticles, 80 ml of acetone were added to precipitate the nanoparticles. The waxy precipitate was recovered by centrifugation. The purification process was repeated two more times to remove excess surfactant and solvent.
정제된 MnO 나노입자(MON)들은 클로로포름에 분산시켜 PEG-포스포리피드 껍질(shell)에 의해 캡슐화되어 생체적합성이 부여되도록 하였다. CHCl3 (5 mg/ml) 내의 2 ml 유기 분산성(organic dispersible) MnO 나노입자들을 10 mg의 1,2-디스테로일-sn-글라이세로-3-포스포에탄올아민-N-[메톡시(폴리에틸렌 글라이콜)-2000]이 포함된 1 ml의 CHCl3 용액과 5:1의 비율로 혼합시켰다. 용매를 증발시킨 후, 80℃에서 1 시간 동안 진공 상태에서 배양하였다. 5 ml의 물을 첨가하여 맑고 짙은 갈색의 현탁액이 생성시켰다. 여과 후, 과잉 mPEG-2000 PE를 초원심분리에 의해 제거되어 정제된 WMON를 수득하였고, 이를 증류수 내에서 분산시킨 후 며칠 동안 저장하였다. Purified MnO nanoparticles (MON) were dispersed in chloroform to be encapsulated by PEG-phospholipid shell to give biocompatibility. 2 ml organic dispersible MnO nanoparticles in CHCl 3 (5 mg / ml) were charged with 10 mg of 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N- [meth Methoxy (polyethylene glycol) -2000] was mixed at a ratio of 5: 1 with 1 ml of CHCl 3 solution. The solvent was evaporated and then incubated at 80 ° C. for 1 hour in vacuo. 5 ml of water were added to give a clear dark brown suspension. After filtration, excess mPEG-2000 PE was removed by ultracentrifugation to obtain purified WMON, which was dispersed in distilled water and stored for several days.
파우더 X-선 회절(XRD) 패턴으로부터 따라 MON 및 WMON의 MnO 주성분을 확인하였다. X-선 광전자 분광기(X-ray Photoelectron Spectroscopy; XPS)를 이용한 표면 조성물 분석에 따라 Mn(II)와 Mn(III)의 존재가 나타났다(도 3). 즉, 합성된 MON는 유기용매하에서 공기와 접촉함으로써 형성된 Mn3O4로 보호막이 씌워지고, 이 들이 물로 이동했을 때 산화가 일어나서 더 두꺼운 Mn3O4 껍질이 형성된 것으로 보인다(도 4). 이와 관련하여, 최근에 공기 하에서의 MnO 표면 산화에 대한 것이 보고된바 있다(A. E. Berkowitz, et al., Phys. Rev.B. 2008, 77, 024403; G. Salazar-Alvarez et al., J. Am. Chem . Soc . 2007, 129, 9102).From the powder X-ray diffraction (XRD) pattern, the MnO principal components of MON and WMON were identified. Surface composition analysis using X-ray photoelectron spectroscopy (XPS) revealed the presence of Mn (II) and Mn (III) (FIG. 3). That is, the synthesized MON is covered with a protective film of Mn 3 O 4 formed by contact with air in an organic solvent, and when they move to water, oxidation occurs and thicker Mn 3 O 4 The shell appears to have formed (FIG. 4). In this regard, it has recently been reported for MnO surface oxidation under air (AE Berkowitz, et al., Phys. Rev. B. 2008, 77, 024403; G. Salazar-Alvarez et al., J. Am . Chem. Soc. 2007, 129 , 9102).
1.3.1.3. HMONHMON 의 합성Synthesis of
도 4에 나타낸 바와 같이, 산성 용액 내 WMON로부터 MnO 페이즈(phase)의 코어를 선택적으로 제거함으로써 HMON의 속이 빈 내부 코어를 제조하였다. 10 mg의 WMON를 20 ml의 4.6 pH 프탈레이트 완충액에 분산시켰고 MnO 코어를 떼어내기 위해 12 시간 동안 교반하였다. 초원심분리에 의해 밝은 갈색 상등액 용액으로부터 HMON를 수득하였으며, 증류수 내 분산과 초원심분리의 반복에 의해 세척하였다As shown in FIG. 4, a hollow inner core of HMON was prepared by selectively removing the core of the MnO phase from WMON in acidic solution. 10 mg of WMON was dispersed in 20 ml of 4.6 pH phthalate buffer and stirred for 12 hours to remove the MnO core. HMON was obtained from a light brown supernatant solution by ultracentrifugation and washed by repetition of dispersion in distilled water and ultracentrifugation.
상기 정제된 HMON 현탁액은 우수한 콜로이드 안정성을 나타내었고, 이로부터 계면활성제 분자에 의한 코팅의 파괴가 없음을 확인하였다. 투과전자현미경 및 주사전자현미경에 의해 내부에 빈 공간을 가지는 HMON의 형성과 합성 초기 물질인 WMON과 비슷한 크기로 분포되어 있는 HMON를 확인하였다(도 5). The purified HMON suspension showed good colloidal stability, from which it was confirmed that there was no breakage of the coating by the surfactant molecule. The transmission electron microscope and the scanning electron microscope confirmed the formation of HMON having an empty space therein and the distribution of HMON having a size similar to that of WMON, which is a synthetic initial material (FIG. 5).
또한, 반복된 실험을 통해서, 산 처리 전에 물(증류수 등)에 WMON을 담그는 시간이 길어질수록 반응산물인 HMON의 내부 모양의 변화가 뚜렷하게 나타나는 것을 관찰하였다(도 6). WMON를 하루 동안 물에 담가 놓았더니 구형과 유사한, 직경 12 nm의 내부의 빈 공간이 생겼고, 물에 담가놓은 후 1일째 HMON의 고해상도 TEM(HRTEM) 이미지에는 다중 결정 부분에 의해 불완전하게 둘러싸인 개방된 구멍이 나타났으며, 물에 담그는 시간이 증가함에 따라 밀폐된 내부 공간이 형성되기 시작하였다. 이에 따라, 물에 담근 후 5일째에 수득한 HMON에서는 폴리크리스탈린 껍질에 의해 둘러싸인 빈 공간이 있는 구조가 형성되었고, 내부 표면에는 주름이 많이 생성되어 HMON 나노 입자의 표면적이 현저하게 증가하였다. HRTEM 분석 결과 물에 10 내지 40일 동안 담가놓은 후 HMON 나노 입자의 평균 직경이 2 내지 20 nm로 나타났고, 이들 구조에는 2 내지 3개의 불규칙한 형태의 구멍이 포함되어 있었다. In addition, through repeated experiments, it was observed that the longer the time of soaking WMON in water (distilled water, etc.) before the acid treatment, the apparent change in the internal shape of the reaction product, HMON, was observed (FIG. 6). Soaking WMON for one day resulted in a hollow interior of 12 nm diameter, similar to a sphere, and on
5일 동안 물에 담가 놓은 후, 제조된 HMON의 XRD 패턴은 이들의 폴리크리스탈린 성질을 반영하여 MnO 부분이 확실하게 감소된 넓은 피크를 나타내었다. 이는 산성 반응 조건에서, 코어에서는 MnO 페이즈의 선택적 용해가 일어나고 껍질을 구성하는 Mn3O4는 저항한다는 것을 나타내는 것이다(도 7a). 또한, 상기 HMON는 실온 부근에서는 상자성인 반면, 저온에서는 강자성을 나타내었는데, 이는 이들의 주성분이 Mn3O4임을 지지하는 결과이다(도 7b). 물에 담가 놓는 시간이 길수록 내부 공간이 줄어드는 것은, 반응물질인 WMON에서 대부분의 MnO 페이즈가 Mn3O4로 전환되기 때문이다. After soaking in water for 5 days, the XRD patterns of the prepared HMON showed broad peaks with a clearly reduced MnO moiety reflecting their polycrystalline properties. This indicates that under acidic reaction conditions, selective dissolution of the MnO phase occurs in the core and the Mn 3 O 4 constituting the shell is resistant (FIG. 7A). In addition, the HMON was paramagnetic at room temperature, while ferromagnetic at low temperature, which is a result of supporting that their main component is Mn 3 O 4 (FIG. 7B). The longer the soak time is, the less the internal space is due to the conversion of most of the MnO phase to Mn 3 O 4 in the reactant WMON.
실시예Example 2. MRI 이완성 측정 2. MRI Relaxation Measurement
본 발명의 MRI 조영제로서의 나노입자의 유효성을 평가하기 위해서, WMON과 WMON을 5일 동안 물에 담근 후에 제조된 HMON의 이완성을 3.0 T 인간 임상 스캐너 를 이용하여 측정하였다. 도 8에 나타난 바와 같이, 동일한 Mn 농도에서 HMON은 T 1- 및 T 2- 효과가 강조된MRI(T 1- and T 2-weighted MRI) 모두에서 WMON 보다 현저하게 증강된 효과를 나타내었다(도 8c). 또한, 조영제의 농도 함수로서 이완율을 측정하여 얻은 특정 이완성은, 고체 상태의 내부를 가지는 WMON의 이완성과 비교하였을 때, HMON에서 현저하게 증강되었다(하기 실시예의 표 1 참고). 이러한 결과는 MnO 코어가 제거됨으로써 새로 형성된 내부 빈 공간의 표면에 Mn의 농도가 증가되어 나타난 것이다. In order to evaluate the effectiveness of the nanoparticles as MRI contrast agent of the present invention, the relaxation of HMON prepared after soaking WMON and WMON for 5 days was measured using a 3.0 T human clinical scanner. As shown in Figure 8, the same amount of Mn is HMON T 1 - and T 2 - the effect is highlighted MRI - exhibited a significantly enhanced effect than WMON in all (T 1 and T 2 -weighted MRI ) ( Fig. 8c ). In addition, the specific relaxation obtained by measuring the relaxation rate as a function of the concentration of the contrast agent was markedly enhanced in HMON compared to the relaxation of WMON having a solid interior (see Table 1 in the Examples below). The result is that the concentration of Mn is increased on the surface of the newly formed internal void space by removing the MnO core.
농도를 변화시키면서 시험관 내에 준비된 WMON과 HMON 각각의 T 1 및 T 2 이완시간은, 80 mT/m의 경사 진폭과 200 ms/m의 슬루율(slew rate)을 갖고 있는 3.0 T 임상용 MRI 스캐너(Philips, Achieva ver. 1.2, Philips Medical Systems, Best, The Netherlands)로 측정되었다. Look-Locker 시퀀스(TR/TE = 10/1 ms 및 flip angle = 5)는 위상 간격 264 ms의 최소 역지연 시간 87 ms를 이용하여 서로 다른 역지연 시간에서의 17 경사 에코 영상들을 얻어내는 방법으로 T 1을 측정하는 데 이용되었다. 여기서, 인-플레인(in-plane) 영상 해상도=625 ?625 mm2이며, 절편 두께=500 mm 이었다. 이 영상들은 3-파라미터 함수에 맞추어져 Matlap 프로그램을 이용하여 T 1 값이 계산되었다. T 2는 다중절편 터보스핀 에코시퀀스 (TR/TE = 5000/20, 40, 60, 80, 100, 120, 140, 160, 180, 200 ms, in-plane 해상도 = 200 ×200 mm2, 절편 두께 = 500 mm)에서의 10 개의 서로 다른 에코 시간들을 이용함으로써 측정되 었다. 이 영상들은 Levenberg-Margardt 방법에 맞추어져 Matlap 프로그램을 이용한 T 2 값이 계산되었다. 조영제의 농도 vs T 1 -1 및 T 2 -1의 플롯(plot) 그래프부터 나노입자들의 특정 이완성(r 1 및 r 2)을 계산하였다. T 1 맵 (60 - 80 pixels) 과T 2 맵 (200 ?00 픽셀)에서 각 ROIs의 신호 강도가 각각의 농도에서 측정되었고, 이것은 각각 r 1 및 r 2 계산에서 이용되었다. ICP-AES에 의해 측정된 망간 원자의 몰 농도 와 산화망간 나노입자 수에 근거하여 두 가지 종류의 특정 이완성을 도출하였다. 산화망간의 나노입자 수에 기초한 이완성은, WMON로서 20 nm 직경의 구형(sphere)에 가까운 것과, 내부 및 외부 직경이 각각 20 nm 및 10 nm인 빈 구멍(sphericla hollow)을 가진 구형에 가까운 것을 가지고 계산되었다. 그 결과를 하기 표 1에 나타내었다.The T 1 and T 2 relaxation times of WMON and HMON prepared in vitro with varying concentrations were 3.0 T clinical MRI scanners (Philips) with a slope amplitude of 80 mT / m and a slew rate of 200 ms / m. , Achieva ver. 1.2, Philips Medical Systems, Best, The Netherlands). The Look-Locker sequence (TR / TE = 10/1 ms and flip angle = 5) is a method of obtaining 17 gradient echo images at different reverse delay times using a minimum reverse delay time of 87 ms with a phase interval of 264 ms. It was used to measure T 1 . In-plane image resolution = 625-625 mm 2 and section thickness = 500 mm. These images were fitted to a three-parameter function and T 1 values were calculated using the Matlap program. T 2 is the multi-section turbospin echosequence (TR / TE = 5000/20, 40, 60, 80, 100, 120, 140, 160, 180, 200 ms, in-plane resolution = 200 × 200 mm 2 , section thickness = 500 mm), using 10 different echo times. These images were fitted to the Levenberg-Margardt method and T 2 values were calculated using the Matlap program. Specific relaxation ( r 1 and r 2 ) of the nanoparticles was calculated from the plot graph of contrast agent vs T 1 -1 and T 2 -1 . The signal intensities of each ROIs in the T 1 map (60-80 pixels) and the T 2 map (200-00 pixels) were measured at the respective concentrations, which were used in the r 1 and r 2 calculations, respectively. Two types of specific relaxation were derived based on the molar concentration of manganese atoms and the number of manganese oxide nanoparticles measured by ICP-AES. Relaxation based on the number of nanoparticles of manganese oxide has WMON as close to a sphere with a diameter of 20 nm and a sphere with sphericla hollow with inner and outer diameters of 20 nm and 10 nm, respectively. Was calculated. The results are shown in Table 1 below.
(ms) T 1 [a]
(ms)
(ms) T 2 [a]
(ms)
(s·mM)-1 r 1
(smM) -1
(s·mM)-1 r 2
(smM) -1
봉입] [b]
(μg)[Dox
Enclosure] [b]
(μg)
(s·μM)-1 r 1
(sμm) -1
(s·μM)-1 r 2
(sμm) -1
봉입] [d]
(μg)[Dox
Enclosure] [d]
(μg)
[a]: 4.5 mM Mn(ICP-AES로 측정됨)에서 측정.[a]: measured at 4.5 mM Mn (measured by ICP-AES).
[b]: Mn 1 mg 당 기준.[b]: Reference per mg Mn.
[c]: 구형의 중공형 모델(spherical hollow model)과 같은 나노입자로부터 얻어진 수치.[c]: Values obtained from nanoparticles such as spherical hollow models.
[d]: 나노입자 1 M 당 기준.[d]: Reference per M of Nanoparticles.
상기 표 1에 나타난 바와 같이, Mn 이온 농도에 기초하여 계산하였을 때, HMON의 r 1 및 r 2는 WMON 보다 각각 7.0 배 및 5.5 배 높았고, 나노입자의 수에 기초하여 계산하였을 때에는 각각 5.8 배 및 4.4 배 더 높았다. As shown in Table 1, when calculated based on the Mn ion concentration, r 1 and r 2 of HMON was 7.0 times and 5.5 times higher than WMON, respectively, 5.8 times and when calculated based on the number of nanoparticles, respectively. 4.4 times higher.
실시예Example 3. 약물 3. Drug 봉입용량Encapsulation Capacity 및 방출 프로파일의 조사 And investigation of emission profiles
본 발명에서는 HMON의 약물 전달 캐리어로서의 성질을 측정하기 위해서, 소수성 항암제인 DOX(doxorubicin)을 모델 항암제로 이용하여 나노입자의 약물 봉입용량(drug-loading capacity)을 확인하였다. In the present invention, in order to measure the properties of HMON as a drug delivery carrier, drug-loading capacity of the nanoparticles was confirmed using a hydrophobic anticancer drug DOX (doxorubicin) as a model anticancer agent.
약물 봉입은, CH3Cl/CH3OH 혼합 용매에서 나노입자와 DOX를 혼합하고, 유기 용매를 증발시킨 후, 물속으로 이동시키는 과정의 프로토콜을 이용하여 수행하였다. 먼저, 나노입자들(WMON 과 HMON)의 결합을 위해, 약물(DOX·HCl)의 하이드로클로라이드염을 소수성 염으로 변환시켰다(Yolles, S. et al. Acta Pharm. Suec. 1978, 15, 382). 소수성 DOX의 제조를 위해 과량의 트리에틸아민(TEA)을 약물(DOX·HCl)의 염화수소 염을 함유하는 메탄올-클로로포름 혼합물 현탁액에 7:1의 비율로 첨가하였다. 수용성 현탁액으로부터 원심분리되어 회수된 고체 HMON(1.57 mg)을 소량의 올레산의 존재 하에서 1 mg 의 DOX 를 포함한 메탄올-클로로포름 혼합물(7 : 1 의 비율) 용액 속으로 분산시켰다. 상기 용매의 증발 후에, 2 ml 물을 첨가하였고, 결과적으로, DOX 가 봉입된 나노입자들 (DOX-WMON 과 DOX-HMON)이 수용성 분산되었다. 도 12a 및12b는 DOX-봉입 HMON를 나타낸 것이고 도 13은 상기 과정을 모식적으로 나타낸 것이다. 봉입되지 않은 DOX는 세 번의 초원심분리 및 증류수 내 재분산을 통해 제거되었다. DOX 봉입을 측정하기 위해, DOX 봉입 나노입자들을 현탁액의 1 ml 분취액의 원심분리에 의해 수집하였고, 메탄올-클로로포름 혼합액 내에서 재분산시켰다. 나노입자들을 원심분리에 의해 용액으로부터 제거하였고, 형광분광광도계(Perkin-Elmer LS 50B)를 이용하여 ex=485 nm와 em=591 nm에서 용액 내 약물 농도를 측정하였다. 표준 플롯 그래프를 나노입자에 봉입된 DOX의 양을 계산하기 위해 동일한 조건에서 작성하였고, 이에 대한 결과를 상기 표 1에 나타내었다.Drug encapsulation was performed using a protocol of mixing nanoparticles and DOX in a CH 3 Cl / CH 3 OH mixed solvent, evaporating the organic solvent, and then moving into water. First, for the binding of nanoparticles (WMON and HMON), the hydrochloride salt of the drug (DOX.HCl) was converted to a hydrophobic salt (Yolles, S. et al. Acta Pharm. Suec. 1978, 15, 382). . Excess triethylamine (TEA) was added in a 7: 1 ratio to the methanol-chloroform mixture suspension containing the hydrogen chloride salt of the drug (DOX.HCl) for the preparation of hydrophobic DOX. Solid HMON (1.57 mg) recovered by centrifugation from an aqueous suspension was dispersed into a methanol-chloroform mixture (7: 1 ratio) solution containing 1 mg of DOX in the presence of a small amount of oleic acid. After evaporation of the solvent, 2 ml water was added and, as a result, DOX-encapsulated nanoparticles (DOX-WMON and DOX-HMON) were water soluble dispersed. 12A and 12B show DOX-enclosed HMON and FIG. 13 schematically illustrates the process. Unsealed DOX was removed through three ultracentrifugation and redispersion in distilled water. To measure DOX inclusion, DOX inclusion nanoparticles were collected by centrifugation of a 1 ml aliquot of the suspension and redispersed in methanol-chloroform mixture. The nanoparticles were removed from the solution by centrifugation and the drug concentration in the solution was measured at ex = 485 nm and em = 591 nm using a fluorescence spectrophotometer (Perkin-Elmer LS 50B). A standard plot graph was drawn under the same conditions to calculate the amount of DOX encapsulated in nanoparticles, and the results are shown in Table 1 above.
상기 표 1에 나타난 바와 같이, 5일 동안 물에 담가 놓은 WMON로 제조한 HMON에 포함된 DOX의 양은, WMON와 비교하였을 때, Mn 이온 농도에 기초한 경우는 HMON가 WMON 보다 4.0 배 더 많고, 나노입자의 수에 기초하였을 때는 HMON가 WMON 보다 3.2 배 더 많은 것으로 나타났다. 이는 DOX 분자가 나노입자 표면을 둘러싸고 있는 올레인산 분자의 소수성 껍질 속으로 분배되어 들어갔기 때문이다(T. K. Jain M., et al., Molecular Pharmaceutics 2005, 2, 194.) 즉, HMON의 높은 봉입 용량은 내부 구멍 안의 주름이 증가하여 HMON의 내부 표면적이 구형의 외부 표면에 비해 증가함으로써 올레인산과 접촉하는 표면적이 증가한 결과이다. As shown in Table 1, the amount of DOX contained in HMON prepared with WMON soaked in water for 5 days was 4.0 times higher than that of WMON based on Mn ion concentration, compared to WMON. Based on the number of particles, HMON was 3.2 times more than WMON. This is because the DOX molecules were distributed into the hydrophobic shell of the oleic acid molecule surrounding the nanoparticle surface (TK Jain M., et al. ,
상기 결과로부터, 본원 발명의 HMON이 암세포를 표적으로 하는 약물 전달체로 사용될 수 있음을 확인하였다. From the above results, it was confirmed that the HMON of the present invention can be used as a drug delivery target for cancer cells.
또한, DOX 방출 프로파일을 수득하기 위하여, 0.1 mg의 DOX 및 0.5 mg의 Mn을 함유하는 DOX-봉입 HMON을 pH 7.4에서 1 mL의 PBS 완충 용액에 분산시키고 실온에서 교반하였다. 0, 1, 2, 4, 12, 24, 60 및 90시간 경과 후, 100 μL의 현탁액을 각각 모아 원심분리하여 나노입자의 고체를 제거하였다. 각각의 시간에 따라 방출된 DOX의 양을 상청액의 형광 강도를 통해 측정하였고 이를 PBS 완충액에서의 표준 곡선과 비교하였다(도 19).In addition, to obtain a DOX release profile, DOX-encapsulated HMON containing 0.1 mg of DOX and 0.5 mg of Mn was dispersed in 1 mL of PBS buffer solution at pH 7.4 and stirred at room temperature. After 0, 1, 2, 4, 12, 24, 60 and 90 hours, 100 μL of suspension was collected and centrifuged to remove the solids of the nanoparticles. The amount of DOX released over each time was measured via the fluorescence intensity of the supernatant and compared with the standard curve in PBS buffer (FIG. 19).
실시예Example 4. 4. HMONHMON 을 이용한 세포 Cell 라벨링Labeling 및 약물 전달 조사 And drug delivery investigation
MR 라벨링(labeling) 및 세포 내 약물 전달자로서의 HMON의 유효성을 DOX-봉입(DOX-loaded) HMON로 인큐베이션된 MCF-7 및MDA-MB-435s 세포를 조사함으로써 평가하였다. The effectiveness of HMON as MR labeling and intracellular drug transporter was assessed by examining MCF-7 and MDA-MB-435s cells incubated with DOX-loaded HMON.
4.1.세포배양 4.1 Cell Culture
인간 유방암세포주, MCF-7 세포들은 가습된 5% CO2 분위기의 37℃에서 성장배지(L-글루타민, 1%항생제-항진균제, 10% 우태아혈청으로 이루어진 RPMI1640)에서 보존되었다. 세포들은 세포성 섭취 실험(cellular uptake experiment) 전에, 24시간 동안 규칙적으로 계대배양 및 자생(reseeded) 되었다. 다른 인간 유방암 세포주, MDA-MB-435s 세포들은 가습된 0% CO2 분위기의 37℃에서 성장배지(L-글루타민, 1% 항생제-항진균제, 10% 우태아 혈청으로 이루어진 L-15)에 보존되었다. 세포들은 세포성 섭취 실험 전에, 24시간 동안 규칙적으로 계대배양 및 자생되었다.Human breast cancer cell line, MCF-7 cells, were conserved in growth medium (RPI1640 consisting of L-glutamine, 1% antibiotic-antifungal, 10% fetal bovine serum) at 37 ° C. in a humidified 5% CO 2 atmosphere. The cells were passaged and reseeded regularly for 24 hours before the cellular uptake experiment. Another human breast cancer cell line, MDA-MB-435s cells, was preserved in growth medium (L-15 consisting of L-glutamine, 1% antibiotic-antifungal, 10% fetal calf serum) at 37 ° C. in a humidified 0% CO 2 atmosphere. . Cells were passaged and autogenated regularly for 24 hours before the cellular uptake experiment.
4.2.4.2. 공초점Confocal 레이저주사현미경( Laser scanning microscope CLSMCLSM ))
MCF-7 및MDA-MB-435s세포들은 세포성 취입 전에 24 시간 동안 챔버 슬라드에서 웰(well)당 5x103 세포의 밀도로 평판 배양되었다. 세포들을 37℃에 2 시간 동안 DOX-HMON에서 배양하였다. 이때 DOX의 농도는 10 μM였다. 이후, 세포들을 차가운 PBS로 세 번 세척하고, 세포핵들은 Hoechst 33342로 염색하였다. 배지에서 10 μM의 최종 농도를 갖는 Hoechst 33342를 세포들에 첨가하고, 세포들을 37℃에서 30분 동안 배양하였다. 배양한 뒤에, 세포들을 4% 파라포름알데하이드 용액에서 고정시켰다. 슬라이드들은 마운팅 배지로 덮었다. 세포들은 Bio-rad사의 라디언스(Radiance) 2100 공초점 현미경 시스템(Confocal Microscope System)을 이용하여 촬영하였다(도 14).MCF-7 and MDA-MB-435s cells were plated at a density of 5 × 10 3 cells per well in chamber slad for 24 hours prior to cellular uptake. Cells were incubated in DOX-HMON at 37 ° C. for 2 hours. At this time, the concentration of DOX was 10 μM. The cells were then washed three times with cold PBS and the nuclei stained with Hoechst 33342. Hoechst 33342 with a final concentration of 10 μM in medium was added to the cells and the cells were incubated at 37 ° C. for 30 minutes. After incubation, cells were fixed in 4% paraformaldehyde solution. Slides were covered with mounting medium. Cells were photographed using a Radiance 2100 Confocal Microscope System from Bio-rad (FIG. 14).
공초점 레이저주사현미경(CLSM)을 통한 관찰 결과, 자유 DOX(free DOX)는 대부분 세포핵에 존재하는 반면에, HMON과 결합된 DOX 분자가 세포질에 있었다. 이는 HMON의 내재화(internalization)가 잘 일어나는 것을 증명하는 것으로, 인큐베이션이 일어나는 2 시간 이내에, 대부분 엔도사이토시스 메커니즘을 통하여, HMON가 DOX를 세포 속으로 운반한 것이다(도 9a 및 9b). Confocal laser scanning microscopy (CLSM) showed that free DOX was mostly present in the cell nucleus, while HMON-associated DOX molecules were in the cytoplasm. This proves that internalization of HMON is well established, within 2 hours of incubation, HMON transported DOX into cells, mostly through endocytosis mechanisms (FIGS. 9A and 9B).
4.3.4.3. DOXDOX , , DOXDOX -봉입 Enclosed HMONHMON 및 And HMONHMON 의 세포독성 평가Cytotoxicity Evaluation of
DOX 봉입 HMON의 시험관 내(in vitro) 세포독성(cytotoxicity)을 MCF-7 세포주에서 측정하고 자유 DOX와 비교하였다. In vitro cytotoxicity of DOX-embedded HMON was measured in the MCF-7 cell line and compared to free DOX.
세포 생존력(viablility)을 37℃ 가습된 5% CO2분위기의 성장배지(L-글루타민, 1% 항생제-항진균제, 10% 우태아 혈청을 포함하는 Leibovitz의 L-15 및 RPMI1640)에 보존된 MCF-7 및 MDA-MB-435s 세포들로 측정하였다. 자유 DOX, 탈 양성자화된 DOX, DOX-봉입 HMON 및 HMON의 세포독성은 MTT 어세이를 이용한 세포성장 억제를 측정하여 평가되었다. 즉, 세포들을 96-웰에 5x103 cell/ 밀도로 평판배양하고, 상기 화합물로 72 시간 동안 처리하였다. 처리가 끝난 뒤, 세포들을 세척하고 MTT 0.5 mg을 첨가하여 3 시간 동안 배양하였다. 세포 생존력은 처리된 세포 수와 비처리된 대조군 세포(non-treated control cells) 수의 비율로 나타냈다. 세포 생존력 그래프를 DOX의 농도에 따라 나타내어, 도 15a(MCF-7) 및 15b(MDA-MB-435s)에 나타내었다. DOX-봉입 HMON의 생존력은 부분적으로는, 배지로 방출된 자유 DOX에 의한 것이다. Cell viability was preserved in MCF- conserved in a growth medium (L-glutamine, 1% antibiotic-antifungal, Leibovitz's L-15 with 10% fetal bovine serum) in a 37% humidified 5% CO 2 atmosphere. 7 and MDA-MB-435s cells were measured. Cytotoxicity of free DOX, deprotonated DOX, DOX-embedded HMON and HMON was assessed by measuring cell growth inhibition using the MTT assay. That is, cells were plated in 96-well at 5 × 10 3 cells / density and treated with the compound for 72 hours. After the treatment, the cells were washed and incubated for 3 hours by adding 0.5 mg of MTT. Cell viability is expressed as the ratio of treated cells to non-treated control cells. Cell viability graphs are shown according to the concentration of DOX and shown in FIGS. 15A (MCF-7) and 15B (MDA-MB-435s). The viability of DOX-enclosed HMON is partly due to free DOX released into the medium.
상기 실험 결과, HMON 단독으로는 실험 조건에서 유효한 세포독성을 보여주지 않았으나, DOX 봉입 HMON은 용량-의존 세포독성 효과를 보여주었고, 이는 동량의 용액 내 DOX와 비교하였을 때 조금 더 낮은 값이었다. 이는 HMON가 가지는 서방형 약물 전달(sustained drug release) 성질 때문인 것으로 보였다. The results showed that HMON alone did not show effective cytotoxicity under experimental conditions, but DOX-embedded HMON showed a dose-dependent cytotoxic effect, which was slightly lower compared to DOX in the same amount of solution. This appears to be due to the sustained drug release nature of HMON.
4.4.세포들의 4.4 Cells in vitroin vitro MRI MRI
1, 10, 50 μg/ml 농도의 DOX-HMON 또는 DOX-WMON으로 배양한 MCF-7 세포들을 원심분리하고, 세포 펠릿들(resulting cell pellets)을 1% 아가로스 용액과 혼합하였다. 상기 혼합물을 MRI촬영을 위해 에펜돌프 튜브(eppendorf tube)로 옮겼다. 아가로스와 세포 혼합물의 T 1- 및 T 2- 효과가 강조된 MRI 촬영을 80 mT/m의 기울기 진폭과 200 ms/m의 슬루율(slew rate)을 갖는, 3.0T 전신 MRI 시스템(Philips, Achieva ver. 1.2, Philips Medical Systems, Best, The Netherlands)을 이용하여 수행하였다(도 9c 및 도 9d).MCF-7 cells incubated with DOX-HMON or DOX-WMON at 1, 10, 50 μg / ml concentration were centrifuged and the resulting cell pellets were mixed with 1% agarose solution. The mixture was transferred to an eppendorf tube for MRI imaging. MRI imaging of the agarose and cell mixture with emphasis on the T 1 -and T 2 -effects was performed using a 3.0T systemic MRI system (Philips, Achieva) with a slope amplitude of 80 mT / m and a slew rate of 200 ms / m. ver. 1.2, Philips Medical Systems, Best, The Netherlands) (FIGS. 9C and 9D).
DOX-봉입 HMON로 처리되지 않은 대조군과 비교하였을 때, DOX-봉입 HMON로 처리된 MCF-7 세포의 MRI 영상에서 농도 의존적 신호 증강(concentration dependent signal enhancement)이 관찰되었고, 유의적인 대비 증강이 T 1-및 T 2-효과가 강조된 영상에서 모두 발견되었다. 도 9c 및 9d에서 보이는 바와 같이, DOX-봉입 HMON가 증가함에 따라 T 1의 효과가 강조된 영상에서의 의미 있는 밝기 대비(brighter contrast)와 T 2의 효과가 강조된 영상에서 의미 있는 어둠 대비(darker contrast)가 관찰되었다. Concentration dependent signal enhancement was observed in MRI images of MCF-7 cells treated with DOX-embedded HMON and compared with controls not treated with DOX-embedded HMON and significant contrast enhancement was T 1. Both-and T 2 -effects were found in the highlighted images. As shown in FIGS. 9C and 9D, as the DOX-encapsulated HMON increases, significant brightness contrast in the image where the effect of T 1 is emphasized and darker contrast in the image where the effect of T 2 is emphasized ) Was observed.
4.5.마우스 4.5.Mouse in in vivovivo MRI MRI
25~35 g의 C3H 마우스 6 마리를 동물 취급에 대한 삼성 바이오메디컬 연구소의 교육 가이드라인 하에 이용하였다. 3 마리에 대해서는, 코어 사이즈가 20 nm인 HMON 1μL(ICP-AES로 측정된 Mn 2.21mg/ml)를 28G 사이즈 바늘을 적용한 정위장치(stereotaxic device)를 이용하여 쥐의 뇌에 주입하였다. HMON의 혈관내 전달을 위하여, 쥐 몸무게 1kg 당 20mg의 Mn(ICP-AES로 측정된)을 쥐의 꼬리 정맥을 통하여 투여하였다. 생체 내(in vivo) MRI 촬영을 위해, 동물들을 마취하고, MR-호환 크래들(cradle)에 놓았다. MRI를 촬영하는 동안, 얼굴 마스크의 산소 과잉 공기를 통해 2% 이소플루랜을 호흡하도록 하여 마우스들을 마취시켰다. 직장의 온도를 주의 깊게 관찰하여 36±1℃로 유지하였다. HMON이 쥐의 몸에 퍼지는 시간을 조사하기 위하여, 3마리 동물에 대하여 HMON을 투여하기 전과 투여 후 3시간, 24시간, 48시간, 72시간 및 7일에 MRI를 촬영하였다(도 10).Six 25-35 g C3H mice were used under Samsung Biomedical Research Institute's educational guidelines for animal handling. For three animals, 1 μL of HMON (2.21 mg / ml Mn measured by ICP-AES) with a core size of 20 nm was injected into the brain of a mouse using a stereotaxic device to which a 28G size needle was applied. For intravascular delivery of HMON, 20 mg of Mn (measured by ICP-AES) per kg of rat weight were administered through the tail vein of the rat. For in vivo MRI imaging, animals were anesthetized and placed in an MR-compatible cradle. During the MRI, mice were anesthetized by having 2% isoflurane breathed through the excess oxygen of the facial mask. The temperature of the rectum was carefully observed and maintained at 36 ± 1 ° C. To investigate the time when HMON spreads in the rat's body, MRIs were taken before and after 3 hours, 24 hours, 48 hours, 72 hours and 7 days of HMON administration for three animals (FIG. 10).
모든 생체 내(in vivo) MRI 촬영은 20 cm 기울기에 100 마이크로초에 400 mT/m 상승시간(rise-time)을 제공할 수 있는, 7T/20 MRI 시스템(Bruker-Biospin, Fallanden, Switzerland)에서 실시되었다. 버드케이지 코일(72mn 내경)(Bruker-Biospin, Fallanden, Swizerland)을 여기(excitation)를 위해 사용하였고, 활동적 분리 상의 어레이 코일을 신호를 받기 위해 사용하였다. 빠른 스핀 반사의 T 1-효과가 강조된 MRI 시퀀스(반복시간(TR)/반사시간(TE)=300/7.9ms, 실험수(NEX)=4, 에코 트레인 길이(echo train length)=2, 800 두께의 슬라이스가 10개 형성된 100x100μm2 플레인 솔루션) 및 빠른 스핀 반사의 T 2-효과가 강조된 MRI 시퀀스(반복시간(TR)/반사시간(TE)=3000/60ms, 실험수(NEX)=4, 에코 트레인 길이(echo train length)=4, 800 두께의 슬라이스가 10개 형성된 100x100μm2 플레인 솔루션)을 사용하여, 고해상도 HMON 명암강화 멀티슬라이스 MR 영상을 각 마우스의 뇌로부터 수득하였다(도 16 내지 도 18).All in vivo MRI imaging was performed on a 7T / 20 MRI system (Bruker-Biospin, Fallanden, Switzerland), which can provide 400 mT / m rise-time at 100 microseconds at 20 cm slope. Was carried out. Birdcage coils (72mn inner diameter) (Bruker-Biospin, Fallanden, Swizerland) were used for excitation and array coils on active separation were used to receive signals. MRI sequence highlighting the T 1 -effect of fast spin reflections (Repeat time (TR) / Reflective time (TE) = 300 / 7.9 ms, Number of experiments (NEX) = 4, Echo train length = 2, 800) 100x100μm 2 plane solution with 10 slices of thickness) and MRI sequence with fast spin reflection T 2 -effect emphasized (repetition time (TR) / reflection time (TE) = 3000 / 60ms, number of experiments (NEX) = 4, Using a high-speed HMON contrast-enhanced multi-slice MR image using echo train length = 4, 800 x 100 μm 2 plane solution with 10 800 thick slices (Figs. 16-18) ).
본 발명의 단순한 변형 내지 변경은 이 분양의 통상의 지식을 가진 자에 의하여 용이하게 실시될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다. Simple modifications and variations of the present invention can be readily made by those skilled in the art, and all such modifications and variations are intended to be included within the scope of this invention.
도 1은 본 발명의 일 실시예에 따른 중공형 나노입자로 이루어진 조영제를 나타낸 그림이다. 1 is a view showing a contrast agent consisting of hollow nanoparticles according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 균일한 산화망간 나노입자(WMnOs)의 제조방법 및 균일한 산화망간 나노입자의 현미경 사진을 나타낸 것이다.Figure 2 shows a micrograph of the manufacturing method of the uniform manganese oxide nanoparticles (WMnOs) and the uniform manganese oxide nanoparticles according to an embodiment of the present invention.
도 3은 본 발명의 일시예에 따른 산화망간 나노입자(MON), WMON, 및 중공형 산화망간 나노입자(hollow manganese oxide nanoparticles; HMON)의 X-선 광전자 분광기(XPS) 그래프를 나타낸 것이다.Figure 3 shows an X-ray photoelectron spectroscopy (XPS) graph of manganese oxide nanoparticles (MON), WMON, and hollow manganese oxide nanoparticles (HMON) according to one embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 HMON을 합성하는 과정으로 모식적으로 나타낸 것이다.4 is a diagram schematically illustrating a process of synthesizing HMON according to an embodiment of the present invention.
도 5는 WMON 및 HMON를 보여주는 투과전자현미경(TEM) 이미지를 나타낸 것이다.5 shows a transmission electron microscope (TEM) image showing WMON and HMON.
도 6은 WMON를 물에 담가 놓는 시간에 따라 HMON의 내부 구조가 영향을 받는다는 것을 보여주는 일련의 TEM 이미지 및 히스토그램으로서, (a)는 WMON를, (b)는 WMON를 하루 동안 물에 담가 놓은 후 제조된 HMON, (c)는 5일, (d)는 10일, (e)는 20일, (f)는 40일 동안 물에 담가 놓은 후 제조된 HMON를 나타낸 것이며, 히스토그램은 나노입자들의 외부 직경(회색 막대)와 내부 공간(흰색 막대)의 크기 분포를 나타내는 것이다.FIG. 6 is a series of TEM images and histograms showing that the internal structure of HMON is affected by the time of soaking WMON, (a) shows WMON and (b) after soaking WMON in water for one day. HMON prepared, (c) is 5 days, (d) is 10 days, (e) is 20 days, (f) is the HMON prepared after soaking in water for 40 days, the histogram is the outside of the nanoparticles It shows the size distribution of the diameter (gray bar) and the inner space (white bar).
도 7의 (a)는 HMON, WMON, MON의 XRD 패턴을 나타낸 것으로서 Mn3O4에 대응하 는 피크는 작은 사각형으로 나타내었고, 그래프 하단의 선들은 큐빅 MnO 페이즈(JCPDS Card. No. 07-0230) 및 테트라고날 Mn3O4 페이즈((JCPDS Card. No. 24-0734)에 해당하는 반사 위치는 나타내는 것이고, 7(b)는 T=5K(점선), T=300K(직선)에서의 MON과 WMON의 히스테리시스 곡선을 나타낸 것이다.Figure 7 (a) shows the XRD patterns of HMON, WMON, MON, the peak corresponding to Mn 3 O 4 is represented by a small square, the bottom line of the graph is a cubic MnO phase (JCPDS Card. No. 07- 0230) and the tetragonal Mn 3 O 4 phase ((JCPDS Card.No. 24-0734) indicates the reflection position, and 7 (b) at T = 5K (dotted line) and T = 300K (straight line). The hysteresis curves of MON and WMON are shown.
도 8의 (a) 및 (b)는 WMON 및 HMON의 농도 vs (a) T 1 -1 및 (b) T 2 -1의 플롯(plot) 그래프이며, (c) 및 (d)는 다양한 Mn 농도에 따른 MON과 WMON 수용성 현탁액에서 얻어진 (c) T 1- 및 (d) T 2-효과가 강조된 MRI이다 8A and 8B are plot graphs of concentrations of WMON and HMON vs. (a) T 1 -1 and (b) T 2 -1 , and (c) and (d) are various Mn MRI emphasizing the (c) T 1 -and (d) T 2 -effects obtained in MON and WMON aqueous suspensions at different concentrations
도 9는, (a) 자유 DOX 및 (b) DOX-봉입 HMON를 이용하여, 10μM DOX 농도에서 2 시간 동안 인큐베이션된 MDA-MB-435s 세포의 DOX 형광의 CLSM 이미지((a)와 (b)의 맨 왼쪽 이미지), Hoechst 33342로 염색된 세포핵 CLSM 이미지 (중간 이미지) 및 통합 이미지를(맨 오른쪽) 나타내고, (c) 및 (d)는 다양한 농도에서 2 시간 동안 인큐베이션된 MCF-7 세포의 (c) T 1-효과가 강조된 MRI 및 (d) T 2-효과가 강조된 MRI이다. 9 shows CLSM images (D) and (b) of DOX fluorescence of MDA-MB-435s cells incubated for 2 hours at 10 μM DOX concentration using (a) free DOX and (b) DOX-encapsulated HMON. Leftmost image), nucleus CLSM image (middle image) and integration image (right image) stained with Hoechst 33342, (c) and (d) of MCF-7 cells incubated for 2 hours at various concentrations ( c) MRI with T 1 -effects highlighted and (d) MRI with T 2 -effects highlighted.
도 10은 국소적으로 HMON가 주입된 마우스 뇌의 (a) T 1-효과가 강조된 MRI 및 (b) T 2-효과가 강조된 MRI이고, (c) HMON을 마우스 혈관 내에 주입한 이후에 마우스 신장의 T 1-효과가 강조된 MRI의 신호 증강의 타임 코스를 나타낸 것이다.10 is of a topically HMON is injected mouse brain (a) T 1 - effect is highlighted MRI and (b) T 2 -, and the effect is highlighted MRI, mouse kidney after injection of (c) HMON in mouse blood The time course of signal augmentation of MRI with the T 1 -effect highlighted is shown.
도 11은, (a) WMON 및 (b) HMON의 주사전자현미경(SEM) 이미지이다.11 is a scanning electron microscope (SEM) image of (a) WMON and (b) HMON.
도 12는, (a) 자연광 하에서의 DOX-봉입 HMON 및 (b) UV(=365 nm) 조사 하에서의 DOX-봉입 HMON 사진을 나타낸 것이다.Figure 12 shows (a) DOX-encapsulated HMON under natural light and (b) DOX-encapsulated HMON under UV (= 365 nm) irradiation.
도 13은 본 발명의 일 실시예에 따른 DOX가 HMON에 봉입되는 과정을 나타내는 모식도이다.13 is a schematic diagram showing a process of encapsulating DOX in HMON according to an embodiment of the present invention.
도 14는 (a) 자유 DOX 및 (b) DOX-봉입 HMON를 이용하여 10 μM DOX 농도에서 2 시간 동안 인큐베이션 된 MCF-7 세포의 DOX 형광 CLSM 이미지((a)와 (b)의 맨 왼쪽 이미지), Hoechst 33342로 염색된 세포핵 CLSM 이미지 (중간 이미지) 및 통합 이미지(맨 오른쪽)를 나타낸 것이다.FIG. 14 shows the leftmost images of DOX fluorescence CLSM images ((a) and (b) of MCF-7 cells incubated for 2 hours at 10 μM DOX concentration using (a) free DOX and (b) DOX-embedded HMON. ), Nucleus CLSM image (middle image) and integration image (rightmost) stained with Hoechst 33342.
도 15는 (a) MCF-7 및(b) MDA-MB-435 세포에 대한 DOX, DOX-HMON, 및 HMON의 세포 독성을 나타내는 그래프이다.FIG. 15 is a graph showing the cytotoxicity of DOX, DOX-HMON, and HMON against (a) MCF-7 and (b) MDA-MB-435 cells.
도 16은 HMON을 마우스 혈관내 주입한 이후에 마우스 신장의 T 1-효과가 강조된 MRI를 시간대 별로 나타낸 것이다.Figure 16 shows the time-dependent MRI highlighting the T 1 -effect of mouse kidney after HMON injection into the mouse vascular.
도 17은 HMON을 마우스 뇌에 혈관내 주입한 이후의 T 1-효과가 강조된MRI를 시간대 별로 나타낸 것이다.Figure 17 shows the time-dependent MRI highlighting the T 1 -effect after intravascular injection of HMON into the mouse brain.
도 18은 HMON을 마우스 뇌에 혈관내 주입한 이후의 T 1- 및 T 2-효과가 동시에 강조된 MRI를 나타낸 것이다.FIG. 18 shows MRI with simultaneous T 1 − and T 2 − effects following endovascular injection of HMON into the mouse brain.
도 19는 PBS 완충 현탁액 내의 DOX-봉입 HMON로부터의 DOX 방출 프로파일을 나타낸다. 19 shows DOX release profile from DOX-encapsulated HMON in PBS buffer suspension.
도 20은 5μM의 Mn 농도에서 DOX-봉입 HMON(좌측) 및 DOX-봉입 WMON(우측)를 이용하여 인큐베이션된 MCF-7 세포(a) 및 MDA-MB-435 세포(b)에 대한 DOX 형광 분광분석 이미지((a)와 (b) 각각의 맨 위쪽), Hoechst 33342로 염색된 세포핵 형광 분광분석 이미지((a)와 (b) 각각의 중간 이미지) 및 통합 이미지((a)와 (b) 각각의 맨 아래쪽)를 나타낸 것이다.FIG. 20 shows DOX fluorescence spectroscopy for MCF-7 cells (a) and MDA-MB-435 cells (b) incubated with DOX-embedded HMON (left) and DOX-embedded WMON (right) at a Mn concentration of 5 μM. Analytical images (top of each of (a) and (b)), nuclear fluorescence spectroscopy images stained with Hoechst 33342 (middle images of (a) and (b) respectively) and integrated images ((a) and (b) Bottom of each one).
도 21은 다양한 농도의 배지에서 2시간 동안 DOX-봉입 HMON 및 DOX-봉입 WMON로 인큐베이션된 MCF-7 세포의 T 1- 및 T 2-효과가 동시에 강조된 MRI 결과를 나타낸 것이다. FIG. 21 shows MRI results simultaneously highlighting the T 1 -and T 2 -effects of MCF-7 cells incubated with DOX-loaded HMON and DOX-loaded WMON for 2 hours in various concentrations of medium.
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