KR20230105122A - Composition comprising cancer cell-target milk extracellular vesicles loaded with anti-cancer drug and use for treatment of cancer - Google Patents
Composition comprising cancer cell-target milk extracellular vesicles loaded with anti-cancer drug and use for treatment of cancer Download PDFInfo
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- KR20230105122A KR20230105122A KR1020220000296A KR20220000296A KR20230105122A KR 20230105122 A KR20230105122 A KR 20230105122A KR 1020220000296 A KR1020220000296 A KR 1020220000296A KR 20220000296 A KR20220000296 A KR 20220000296A KR 20230105122 A KR20230105122 A KR 20230105122A
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- milk
- extracellular vesicles
- cancer
- derived extracellular
- oxaliplatin
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Abstract
본 발명은 우유에서 추출한 세포외소포체에 관한 것으로, 보다 상세하게는 암세포를 표적할 수 있는 GE11이 표지된 항암 약물 옥살리플라틴 로딩 우유 세포외소포체의 약제학적 조성물 및 이의 제조방법에 그리고 암 세포의 생존 억제 및 세포 자연사 유도 방법을 제공하는 것이다. 본 발명에 따른 GE11 표지 옥살리플라틴 로딩 우유 세포외소포체는 대조군 우유 세포외소포체 대비 인간 결장암 세포 (SNU-C5) 그리고 인간 유방암 세포(MDA-MB-231)에 높은 효율로 표적화 할 수 있으며, 항암제인 옥살리플라틴을 부착된 세포에 특이적으로 전달할 수 있다.The present invention relates to extracellular vesicles extracted from milk, and more particularly, to a pharmaceutical composition of GE11-labeled anticancer drug oxaliplatin-loaded milk extracellular vesicles capable of targeting cancer cells, a method for preparing the same, and inhibition of cancer cell survival And to provide a method for inducing apoptosis. The GE11-labeled oxaliplatin-loaded milk extracellular vesicles according to the present invention can target human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231) with high efficiency compared to control milk extracellular vesicles, and the anticancer drug oxaliplatin can be delivered specifically to attached cells.
Description
본 발명은 우유에서 유래된 세포외소포체에 관한 것으로, 보다 상세하게는 암세포를 표적하는 항암약물이 로딩된 우유 유래 세포외소포체의 약제학적 조성물 및 이의 제조방법을 제공한다.The present invention relates to milk-derived extracellular vesicles, and more particularly, provides a pharmaceutical composition of milk-derived extracellular vesicles loaded with an anticancer drug targeting cancer cells and a method for preparing the same.
암은 비정상적인 세포 성장으로 인하여 유발되는 질병으로 신체의 다른 장기를 침범하거나 퍼질 가능성을 지니고 있는 악성 종양을 말한다. 암세포는 성장인자가 공급되지 않더라도 자기 스스로 공급하거나 성장할 수 있으며, 성장 억제 신호에도 저항성을 가지게 되어 지속적인 성장과 분열이 가능한 특징을 가졌다. 또한 정상적인 세포사멸 과정에 대한 저항성 및 회피 능력을 가지고 있어 쉽게 제거되지 않으며 무한한 세포 분열이 가능하다. 그리고 주위의 혈관 생성을 촉진시키는 능력을 이용하여 암세포 주변으로 혈관을 유도하여 끊임없이 영양분을 공급하여 생존에 유리하게 된다. Cancer is a disease caused by abnormal cell growth, and refers to a malignant tumor that has the potential to invade or spread to other organs in the body. Cancer cells can supply or grow on their own even if growth factors are not supplied, and have resistance to growth inhibitory signals, so they have the characteristics of being able to continuously grow and divide. In addition, it has the ability to resist and evade the normal cell death process, so it is not easily removed and infinite cell division is possible. In addition, by using the ability to promote the generation of surrounding blood vessels, blood vessels are induced around the cancer cells to continuously supply nutrients, which is advantageous for survival.
현재까지 암은 우리나라의 사망률 1위를 차지하고 있는데, 그 중 대장암 (Colorectal cancer)은 전체 암 중 발생 빈도 3위를 차지하는 호발 암으로 연간 암 발생 증가율은 6.7 %에 달한다. 특히 대장암은 높은 암 발생과 더불어 높은 사망률을 지니고 있는데, 전 세계적으로 매년 608,000명의 환자가 대장암으로 사망하는 것으로 추정되고 있다. 이는 전체 암 환자 사망의 8 %를 차지하고, 암으로 인한 환자 사망 중 4위를 차지하고 있다. 국내 기준으로는 암 사망률 중 대장암이 3위를 차지하고 있다. 10년 전과 비교하면 암 사망률이 약 22.1 % 증가하였고, 통계에 따르면 2030년 국내 발병 건수가 2배 이상 급증할 것으로 추정되고 있다. 대장암의 경우 종양의 크기가 아니라 종양의 조직 침투 정도에 따라 치료 방법을 결정하며 현재 치료 방법으로는 수술, 항암화학요법, 방사선 치료를 함께 병행하는 것을 원칙으로 하고 있다. 절제 수술 후 보조적 항암치료 (항암 화학요법)까지 병용 치료를 통해 대장암의 5년 생존율은 증가했으나 25 내지 35 %의 환자들에게서 전이가 나타나며, 암 환자 중 약 20 %에서는 대장암 진단 당시 타 장기로의 전이가 동시에 진단되며, 전이성 대장암 환자와 재발된 암환자에게 항암약물치료가 주된 치료가 되지만 완치가 어렵다고 보고 되어 있다. 또한, 대장암 말기에는 암의 줄기세포가 간 (40 %), 폐 (15 %), 복막 (28 %), 뼈 (16 %), 부신(14 %), 난소 (18 %) 등 절제술이 어려운 곳으로 전이되어 주요 사망원인이 되고 있다. 이러한 이유로 대장암의 진행 및 전이 억제에 대한 새로운 치료제 개발 연구가 시급한 실정이다. Until now, cancer occupies the first place in Korea's mortality rate, and among them, colorectal cancer is a prevalent cancer that ranks third among all cancers, with an annual cancer incidence rate of 6.7%. In particular, colorectal cancer has a high mortality rate along with a high incidence of cancer, and it is estimated that 608,000 patients worldwide die from colorectal cancer every year. It accounts for 8% of all cancer patient deaths and ranks fourth among cancer patient deaths. Colorectal cancer ranks third among cancer mortality rates in Korea. Compared to 10 years ago, the cancer death rate increased by about 22.1%, and according to statistics, it is estimated that the number of domestic outbreaks will more than double by 2030. In the case of colorectal cancer, the treatment method is determined not by the size of the tumor but by the degree of tissue penetration of the tumor, and the current treatment method is to combine surgery, chemotherapy, and radiation therapy in principle. Although the 5-year survival rate of colorectal cancer increased through combination treatment from resection surgery to adjuvant chemotherapy (chemotherapy), 25 to 35% of patients develop metastases, and about 20% of cancer patients have other organs at the time of diagnosis of colorectal cancer. Metastasis is diagnosed at the same time, and chemotherapy is the main treatment for metastatic colorectal cancer patients and recurrent cancer patients, but it has been reported that a complete cure is difficult. In addition, in the late stage of colorectal cancer, stem cells of cancer are transferred to liver (40%), lung (15%), peritoneum (28%), bone (16%), adrenal gland (14%), and ovary (18%), where resection is difficult. It has metastasized and is a leading cause of death. For this reason, there is an urgent need for research on the development of new therapeutic agents for suppression of progression and metastasis of colorectal cancer.
대장암 치료 방법으로 전이의 발생을 줄이고, 종양 크기를 감소시키거나 종양의 성장을 느리게 하기 위하여 항암화학요법이 흔히 사용되고 있다. 특히 항암화학 요법 중 FOLFOX4는 대장암의 stage Ⅲ 및 Ⅳ의 표준치료법으로 사용되고 있는데, 이는 옥살리플라틴을 기초로 하는 항암화학요법으로 알려져 있다. 옥살리플라틴을 기초로 하는 항암화학요법은 3기 대장암 환자의 3년 간 무병 생존율을 78.2 %로 향상 시킬 정도로 치료 효과가 높다. 이러한 이유로 1차 치료제로 선택되는 보조항암화학약제로 흔히 5-플루오로우라실 (5-Fluorouracil), 류코보린 (Leucovorin)과 함께 사용되고 있으며, 이는 FOLFOX 요법으로 알려져 있다. 옥살리플라틴은 근래에 개발된 3세대 플라틴 (platin)의 유사체로 알려져 있지만, 그 효과와 부작용은 시스플리틴 (cisplatin)과 다르다고 보고되고 있다. 옥살리플라틴은 신독성 및 이독성이 적은 편이며, 시스플라틴 저항성 대장암 세포에서도 효과가 있다고 보고되었다. 옥살리플라틴은 시스플라틴과 유사한 기전으로 작용하는데, 그 기전을 살펴보면 암세포의 DNA 가닥 사이에 다리를 연결하는 platinum-DNA crosslink를 형성하여 암세포의 DNA 합성을 방해하여 항암효과를 일으킨다고 보고되었다. 더구나 옥살리플라틴은 5-플루오로우라실과 함께 사용할 경우 그 효과가 높아지는 것으로 보고되었고, 실제 환자를 대상으로 한 임상 실험에서도 5-플루오로우라실 단독요법에 비해 옥살리플라틴을 함께 사용 할 경우 효과가 탁월하게 증가함이 증명되었다. 이러한 이유로 재발을 하거나 전이가 되어 완전 절제가 어려운 대장암 환자에서 5-플루오로우라실과 더불어 옥살리플라틴을 병행하여 사용하므로 환자의 생존율은 증가되었다. 하지만, 옥살리플라틴에게도 용량 제한 독성인 말초신경병증 (85 내지 95 %), 알라닌아미노전이효소 증가 (54 %), 아스파테이트아미노전이효소 증가 (36 % grade 3/4: 1 %), 혈소판 감소증 (30 %), 설사 (46 %), 메스꺼움 (64 %), 구토 (37 %) 등의 부작용이 보고되고 있다. 이러한 부작용은 다양한 이유에서 나타날 수 있지만, 표적인 암세포 외 다른 정상적인 세포에도 영향을 주기 때문에 일어난다고 여겨진다. 그러기에 이러한 항암 약물을 암세포에만 표적 할 수 있는 새로운 치료 방법이 필요하다. As a method of treating colorectal cancer, chemotherapy is commonly used to reduce the occurrence of metastasis, reduce tumor size, or slow tumor growth. Particularly, among chemotherapy, FOLFOX4 is used as a standard treatment for stages Ⅲ and Ⅳ of colorectal cancer, which is known as oxaliplatin-based chemotherapy. Chemotherapy based on oxaliplatin is highly effective enough to improve the 3-year disease-free survival rate of patients with
이러한 약물을 암세포에게만 전달하기 위한 전달체 연구로 합성 나노입자나 세포 유래 세포외소포체 등 다양한 연구들이 진행되고 있다. 하지만 기존 나노입자 전달체의 경우 금, 무기물질 등과 같은 입자를 기반으로 하여 생체적합성과 생체 내 분해성이 떨어져 인체에 적용하는데 어려움이 있다. 또한 세포 유래 세포외소포체는 생체적합성 및 생분해성이 높아 인체 적용에 유리한 반면에 대량 생산 및 안정성이 낮아 약물을 전달하기 위한 전달체로 사용하는데 한계점이 있다. 그러기에 이러한 제한점을 극복하기 위해 다양한 연구가 진행되고 있고, 최근 우유 세포외소포체가 그 대안점으로 떠오르고 있다. 우유 세포외소포체는 생물학적 물질들로 이루어져 있기 때문에 생체적합성과 생분해성이 높으며, 체내 면역반응도가 낮은 안전한 전달체이다. 또한 충분한 양의 세포 배양액을 얻기 위해서는 많은 시간과 비용이 필요한 것에 비해, 우유는 적은 시간과 비용으로 간편하게 확보할 수 있는 이점이 있다. 우유 세포외소포체는 대량 생산이 가능하다고 알려져 있는데, 우유 세포외소포체는 통상적으로 세포 배양액에서 얻을 수 있는 세포외소포체에 비하여 단백질 양으로는 65,483배, particle 양으로는 860배 더 많은 양을 얻을 수 있다. 따라서 우유 세포외소포체를 이용한 항암 물질 전달체의 개발이 임상 진입에 용이하다고 할 수 있다는 것을 의미한다. 더욱이 우유 세포외소포체는 소수성 또는 친수성 약물 모두 로딩이 가능하며, 전기천공법 또는 화학형질주입법을 이용하여 다양한 유전자 로딩이 가능한 이점이 있으며, 우유 세포외소포체는 기존 세포 유래 세포외소포체에 사용되는 다양한 표면 개질화 (surface modification)방법을 적용하여 표적성 물질이 부착된 표적 특이적 전달체 개발이 용이하다. 즉, 우유 엑소좀을 이용한 암 세포 표적화가 가능함을 의미한다. 이러한 이유로 암세포 표적화가 가능한 우유 엑소좀에 항암 약물을 로딩한다면, 인체에 유해하지 않으며 부작용은 적은 항암제로 사용 가능할 것이라 판단되어 이에 대한 연구가 계속 요구되고 있다. As a delivery vehicle for delivering these drugs only to cancer cells, various studies such as synthetic nanoparticles or cell-derived extracellular vesicles are being conducted. However, existing nanoparticle delivery systems are based on particles such as gold and inorganic materials, and have poor biocompatibility and biodegradability, making it difficult to apply them to the human body. In addition, cell-derived extracellular vesicles are advantageous for human application due to their high biocompatibility and biodegradability, but have limitations in their use as a delivery system for drug delivery due to low mass production and stability. Therefore, various studies are being conducted to overcome these limitations, and milk extracellular vesicles have recently emerged as an alternative. Because milk extracellular vesicles are composed of biological materials, they are highly biocompatible and biodegradable, and are safe delivery vehicles with low immune reactivity in the body. In addition, compared to requiring a lot of time and money to obtain a sufficient amount of cell culture medium, milk has the advantage of being easy to obtain with little time and money. It is known that milk extracellular vesicles can be mass-produced, and milk extracellular vesicles can be obtained in 65,483 times more protein and 860 times more particles than the extracellular vesicles normally obtained from cell culture. there is. Therefore, it means that the development of an anti-cancer substance delivery system using milk extracellular vesicles can be said to be easy for clinical entry. Furthermore, milk extracellular vesicles can be loaded with either hydrophobic or hydrophilic drugs, and have the advantage of being able to load various genes using electroporation or chemical transfection. It is easy to develop a target-specific carrier to which a target substance is attached by applying a surface modification method. That is, it means that cancer cell targeting using milk exosomes is possible. For this reason, if an anticancer drug is loaded into milk exosomes capable of targeting cancer cells, it is judged that it can be used as an anticancer drug that is not harmful to the human body and has few side effects, and research on this is continuously required.
본 발명의 목적은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 유래 세포외소포체를 포함하는 암 질환 예방 또는 치료용 약학 조성물 및 이의 제조방법을 제공하는 데에 있다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer disease comprising milk-derived extracellular vesicles labeled with a GE11 peptide and loaded with an anticancer agent, and a method for preparing the same.
본 발명의 또 다른 목적은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 세포외소포체를 포함하는 암 질환 예방 또는 개선용 건강식품용 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a health food composition for preventing or improving cancer disease, comprising milk extracellular vesicles labeled with the GE11 peptide and loaded with an anticancer agent.
상기 목적을 달성하기 위하여, 본 발명은 GE11 펩티드가 표지된 우유 유래 세포외소포체를 포함하는 암 세포 표적형 약물전달용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for cancer cell-targeted drug delivery comprising milk-derived extracellular vesicles labeled with the GE11 peptide.
또한, 본 발명은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 유래 세포외소포체를 포함하는 암 질환 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer disease comprising milk-derived extracellular vesicles labeled with the GE11 peptide and loaded with an anticancer agent.
또한, 본 발명은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 세포외소포체를 포함하는 암 질환 예방 또는 개선용 건강식품용 조성물을 제공한다.In addition, the present invention provides a health food composition for preventing or improving cancer disease, comprising milk extracellular vesicles labeled with the GE11 peptide and loaded with an anticancer agent.
또한, 본 발명은 a) 우유를 원심분리하여 우유 유래 세포외소포체를 분리하는 단계; b) 상기 a) 단계에서 수득된 우유 유래 세포외소포체를 콜레스테롤-폴리에틸렌글리콜(PEG)-DBCO에 혼합한 후, GE11 펩티드에 혼합하여 GE11 표지를 부착하는 단계; 및 c) 상기 b) 단계에서 수득된 GE11 표지 우유 유래 세포외소포체를 항암약물과 인큐베이션하는 단계;를 포함하는 상기 약학 조성물 제조방법을 제공한다.In addition, the present invention a) separating the milk-derived extracellular vesicles by centrifuging the milk; b) mixing the milk-derived extracellular vesicles obtained in step a) with cholesterol-polyethylene glycol (PEG)-DBCO, and then mixing with GE11 peptide to attach a GE11 label; and c) incubating the GE11-labeled milk-derived extracellular vesicles obtained in step b) with an anticancer drug.
본 발명에 따른 GE11이 부착된 옥살리플라틴 로딩 우유 세포외소포체는 대조군 우유 세포외소포체 대비 인간 결장암 세포주 (SNU-C5) 및 인간 유방암 세포주 (MDA-MB-231)에 높은 효율로 부착할 수 있으며, 항암제인 옥살리플라틴을 부착된 세포에 특이적으로 전달할 수 있어, 인간 결장암 세포 (SNU-C5) 및 인간 유방암 세포 (MDA-MB-231)의 생존율을 떨어트리고, 세포 자멸사를 유도하는 효과가 있는 것을 확인하여, 본 발명에 따른 GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 이의 제조방법은 항암 치료 분야에서 다양하게 활용될 수 있다.Oxaliplatin-loaded milk extracellular vesicles attached with GE11 according to the present invention can attach to human colon cancer cell line (SNU-C5) and human breast cancer cell line (MDA-MB-231) with high efficiency compared to control milk extracellular vesicles, and are anticancer agents Phosphorus oxaliplatin can be delivered specifically to attached cells, reducing the viability of human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231) and inducing apoptosis. , The GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular cell body according to the present invention and the preparation method thereof can be variously utilized in the field of anti-cancer treatment.
도 1에서, 도 1A는 우유 유래 세포외소포체와 293T세포에서 유래한 세포외소포체를 고온 처리 (75 ℃) 하고 세포외소포체 마커 CD9의 발현을 분석한 결과; 도 1B는 우유 유래 세포외소포체와 293T세포에서 유래한 세포외소포체를 고온 처리 (75 ℃) 하고 단백질의 농도를 정량화한 결과; 도 1C는 우유 유래 세포외소포체와 293T세포에서 유래한 세포외소포체를 고온 처리 (75 ℃) 하고 입자의 농도를 정량화한 결과; 도 1D는 우유 유래 세포외소포체와 293T세포에서 유래한 세포외소포체를 중성 (pH 7.2) 또는 낮은 pH (pH 5) 조건에 노출된 후 세포외소포체 마커 CD9의 발현을 분석한 결과; 도 1E는 우유에서 유래한 세포외소포체와 293T세포에서 유래한 세포외소포체를 중성 (pH 7.2) 또는 낮은 pH (pH 5) 조건에 노출된 후 단백질의 농도를 정량화한 결과; 및 도 1F는 우유 유래 세포외소포체와 293T세포에서 유래한 세포외소포체를 중성 (pH 7.2) 또는 낮은 pH (pH 5) 조건에 노출된 후 입자의 농도를 정량화한 결과;를 나타낸다.
도 2에서, 도 2A는 우유 유래 세포외소포체 및 콜레스테롤-폴리에틸렌글리콜(PEG)-비오틴(biotin) (이하, 콜레스테롤-PEG-비오틴이라함)이 반응하여 세포외소포체에 콜레스테롤-PEG-비오틴이 부착한 것을 도트 블롯 분석을 통하여 확인한 결과; 도 2B는 우유 유래 세포외소포체와 콜레스테롤-PEG-비오틴의 최적 혼합 농도를 보여주는 도트 블롯 분석 결과; 및 도 2C는 우유 유래 세포외소포체와 콜레스테롤-PEG-비오틴의 최적 혼합 농도를 보여주는 정량화 결과;를 나타낸다.
도 3에서, 도 3A는 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 옥살리플라틴을 로딩한 후 전자현미경으로 세포외소포체들의 외형을 관찰한 결과; 도 3B는 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 옥살리플라틴을 로딩한 후 나노입자추적분석법 (nanoparticle tracking analysis)을 통하여 세포외소포체의 크기를 확인한 결과; 도 3C는 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 옥살리플라틴을 로딩한 후 나노입자추적분석법 (nanoparticle tracking analysis)을 통하여 세포외소포체의 크기의 평균을 확인한 결과; 및 도 3D는 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 옥살리플라틴을 로딩한 후 웨스턴 블롯을 통하여 세포외소포체 마커를 확인한 결과;를 나타낸다.
도 4에서, 도 4A는 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 웨스턴 블롯을 통하여 GE11 마커를 확인한 결과; 도 4B는 250, 500 및 1000 μg/mL의 옥살리플라틴이 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 혼합 되었을 때, 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체의 옥살리플라틴 로딩 정량화 결과; 및 도 4C는 PBS에서 최대 36시간 동안 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에서의 옥살리플라틴 방출 결과;를 나타낸다.
도 5에서, 도 5A는 인간 제대 정맥 내피 세포 (HUVEC), 인간 결장암 세포 (SNU-C5) 및 인간 유방암 세포(MDA-MB-231)에서 EGRF에 대한 웨스턴 블롯 분석 결과; 도 5B는 인간 제대 정맥 내피 세포 (HUVEC), 인간 결장암 세포 (SNU-C5) 및 인간 유방암 세포 (MDA-MB-231)에 CMO가 표지된 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 처리한 후, 세포의 유세포 분석을 한 결과; 및 도 5C 및 5D는 인간 제대 정맥 내피 세포 (HUVEC), 인간 결장암 세포 (SNU-C5) 그리고 인간 유방암 세포 (MDA-MB-231)에 CMO가 표지된 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 처리한 후 공초점 현미경 이미지 결과 (스케일 막대 = 40 μm);를 나타낸다.
도 6에서, 도 6A는 인간 결장암 세포 (SNU-C5)에 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 옥살리플라틴을 처리한 후, 세포의 생존률을 확인한 결과; 도 6B는 인간 유방암 세포 (MDA-MB-231)에 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 옥살리플라틴을 처리한 후, 세포의 생존률을 확인한 결과; 도 6C는 인간 결장암 세포 (SNU-C5) 및 인간 유방암 세포 (MDA-MB-231)에 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 옥살리플라틴을 처리한 후, 세포를 annexin V-fluorescein isothiocyanate (FITC) 및 propidium iodide (PI)(Sigma Aldrich)로 염색하여 유세포 분석을 통하여 세포자멸사를 분석한 결과; 도 6D는 인간 결장암 세포 (SNU-C5)에 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 옥살리플라틴을 처리한 후, 세포를 annexin V-fluorescein isothiocyanate (FITC) 및 propidium iodide (PI)(Sigma Aldrich)로 염색하여 유세포 분석을 통하여 세포자멸사를 분석하여 정량화한 결과; 및 도 6E은 인간 유방암 세포 (MDA-MB-231)에 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 및 옥살리플라틴을 처리한 후, 세포를 annexin V-fluorescein isothiocyanate (FITC) 및 propidium iodide (PI)(Sigma Aldrich)로 염색하여 유세포 분석을 통하여 세포자멸사를 분석하여 정량화한 결과;를 나타낸다.
도 7은 종양이식 대장암 종양 동물 모델에 GE11 펩티드가 표지된 우유 유래 세포외 소포체에 옥살리플라틴을 1, 2 및 5 mg/kg 적용한 후, 항종양 효과를 분석한 결과를 나타낸다.
도 8에서, 도 8A는 종양이식 대장암 종양 동물 모델에 PBS, 옥살리플라틴 로딩 우유 유래 세포외소포체 (5 mg/kg), 옥살리플라틴 (5 mg/kg), 옥살리플라틴 (20 mg/kg) 및 GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 (5 mg/kg)를 처리한 후, 종양의 크기를 확인한 결과; 및 도 8B는 종양이식 대장암 종양 동물 모델에 PBS, 옥살리플라틴 로딩 우유 유래 세포외소포체 (5 mg/kg), 옥살리플라틴 (5 mg/kg), 옥살리플라틴 (20 mg/kg) 및 GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 (5 mg/kg)를 처리한 후, 종양의 크기를 정량화한 결과;를 나타낸다.In Figure 1, Figure 1A is a result of high temperature treatment (75 ℃) of milk-derived extracellular vesicles and extracellular vesicles derived from 293T cells and analyzing the expression of the extracellular vesicle marker CD9; Figure 1B is a result of high-temperature treatment (75 ° C.) of milk-derived extracellular vesicles and 293T cell-derived extracellular vesicles and quantification of protein concentration; Figure 1C is a result of high temperature treatment (75 ° C.) of milk-derived extracellular vesicles and 293T cell-derived extracellular vesicles and quantification of particle concentration; Figure 1D is a result of analyzing the expression of the extracellular vesicle marker CD9 after exposing milk-derived extracellular vesicles and extracellular vesicles derived from 293T cells to neutral (pH 7.2) or low pH (pH 5) conditions; Figure 1E is a result of quantifying the protein concentration after exposing milk-derived extracellular vesicles and 293T cell-derived extracellular vesicles to neutral (pH 7.2) or low pH (pH 5) conditions; And Figure 1F shows the result of quantifying the concentration of particles after exposing milk-derived extracellular vesicles and extracellular vesicles derived from 293T cells to neutral (pH 7.2) or low pH (pH 5) conditions.
In FIG. 2, FIG. 2A shows milk-derived extracellular vesicles and cholesterol-polyethylene glycol (PEG)-biotin (hereinafter referred to as cholesterol-PEG-biotin) react to attach cholesterol-PEG-biotin to the extracellular vesicles. The result confirmed through dot blot analysis; Figure 2B is a result of dot blot analysis showing the optimal mixing concentration of milk-derived extracellular vesicles and cholesterol-PEG-biotin; And Figure 2C shows the quantification results showing the optimal mixing concentration of milk-derived extracellular vesicles and cholesterol-PEG-biotin.
In Figure 3, Figure 3A is a result of observing the appearance of the milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles after loading oxaliplatin and then electron microscopy; Figure 3B is a result of confirming the size of extracellular vesicles through nanoparticle tracking analysis after loading oxaliplatin on milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles; Figure 3C is a result of confirming the average size of extracellular vesicles through nanoparticle tracking analysis after loading oxaliplatin on milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles; And Figure 3D shows the results of confirming the extracellular vesicle markers through Western blot after loading oxaliplatin on milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles.
In FIG. 4, FIG. 4A shows the result of confirming the GE11 marker through Western blot for milk-derived extracellular vesicles loaded with oxaliplatin and milk-derived extracellular vesicles labeled with the GE11 peptide; Figure 4B shows that when 250, 500 and 1000 μg/mL of oxaliplatin were mixed with milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles, milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular quantification of oxaliplatin loading in the endoplasmic reticulum; And Figure 4C shows the results of oxaliplatin release from milk-derived extracellular vesicles loaded with oxaliplatin and GE11 peptide-labeled milk-derived extracellular vesicles for up to 36 hours in PBS.
In FIG. 5, FIG. 5A shows the results of Western blot analysis for EGRF in human umbilical vein endothelial cells (HUVEC), human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231); 5B shows human umbilical vein endothelial cells (HUVEC), human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231) labeled with milk-derived extracellular vesicles and GE11 peptide loaded with CMO-labeled oxaliplatin. After processing the milk-derived extracellular vesicles, the result of flow cytometry analysis of the cells; And Figures 5C and 5D show human umbilical vein endothelial cells (HUVEC), human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231) loaded with CMO-labeled oxaliplatin, milk-derived extracellular vesicles and GE11 Confocal microscopy image results after processing the peptide-labeled milk-derived extracellular vesicles (scale bar = 40 μm); are shown.
In Figure 6, Figure 6A shows human colon cancer cells (SNU-C5) milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, GE11 peptide-labeled oxaliplatin-loaded milk-derived After treating the extracellular cell body and oxaliplatin, the result of confirming the cell viability; Figure 6B shows milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, and GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular cells in human breast cancer cells (MDA-MB-231). After treatment with the cell body and oxaliplatin, the result of confirming the viability of the cells; 6C shows milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, and GE11 peptides in human colon cancer cells (SNU-C5) and human breast cancer cells (MDA-MB-231). After treatment with labeled oxaliplatin-loaded milk-derived extracellular cell bodies and oxaliplatin, the cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Sigma Aldrich), and apoptosis was analyzed through flow cytometry. ; Figure 6D shows human colon cancer cells (SNU-C5) milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular cells, and After treatment with oxaliplatin, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Sigma Aldrich), and apoptosis was analyzed and quantified through flow cytometry; And Figure 6E shows human breast cancer cells (MDA-MB-231) milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, GE11 peptide-labeled oxaliplatin-loaded milk-derived cells After treatment with exocytosis and oxaliplatin, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Sigma Aldrich), and apoptosis was analyzed and quantified through flow cytometry;
7 shows the results of analyzing the antitumor effect after applying 1, 2, and 5 mg/kg of oxaliplatin to milk-derived extracellular vesicles labeled with the GE11 peptide in a tumor-transplanted colorectal cancer tumor animal model.
In FIG. 8, FIG. 8A shows PBS, oxaliplatin-loaded milk-derived extracellular vesicles (5 mg/kg), oxaliplatin (5 mg/kg), oxaliplatin (20 mg/kg), and GE11 peptide in a tumor transplantation colorectal cancer tumor animal model. After processing the labeled oxaliplatin-loaded milk-derived extracellular cell body (5 mg/kg), the size of the tumor was confirmed; and Figure 8B shows PBS, oxaliplatin-loaded milk-derived extracellular vesicles (5 mg/kg), oxaliplatin (5 mg/kg), oxaliplatin (20 mg/kg), and GE11 peptide-labeled oxaliplatin in a tumor transplantation colon cancer tumor animal model. The result of quantifying the size of the tumor after processing the loaded milk-derived extracellular cell body (5 mg/kg); is shown.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
항암제는 암 세포 뿐만 아니라, 다른 정상적인 세포에도 영향을 주기 때문에 이로 인하여 발생하는 부작용을 줄이기 위해, 암 세포에 표적을 위해 우유 유래 세포외소포체를 포함하는 본 발명을 완성하였다.Since the anticancer agent affects not only cancer cells but also other normal cells, in order to reduce the side effects caused by this, the present invention including milk-derived extracellular vesicles for targeting to cancer cells was completed.
본 발명은 GE11 펩티드가 표지된 우유 유래 세포외소포체를 포함하는 암 세포 표적형 약물전달용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for cancer cell-targeted drug delivery comprising milk-derived extracellular vesicles labeled with a GE11 peptide.
상기“세포외소포체 (extracellular vesicle, EV)”는 다양한 세포들로부터 분비되는 막 구조의 작은 (30 내지 1000 nm의 직경) 소낭을 의미하며, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. The “extracellular vesicle (EV)” refers to a small (diameter of 30 to 1000 nm) vesicle with a membrane structure secreted from various cells, and is released into the extracellular environment by fusion of the polycystic body and the plasma membrane. means vesicle.
또한, 본 발명은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 유래 세포외소포체를 포함하는 암 질환 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer disease comprising milk-derived extracellular vesicles labeled with the GE11 peptide and loaded with an anticancer agent.
상기 우유 유래 세포외소포체는 우유를 원심분리하여 수득할 수 있는데, 바람직하게 우유를 1,600 내지 2,400 g에서 5 내지 15분 및 그 후에 7,000 내지 13,000 g에서 30 내지 50분 동안 1차 원심분리하고, 상기 1차 원심분리로 수득한 상층액을 30,000 내지 40,000 g에서 40 내지 80분간 2차 원심분리하며, 상기 2차 원심분리로 수득한 상층액을 50,000 내지 150,000 g에서 40 내지 80분 동안 3차 원심분리하여 수득될 수 있다.The milk-derived extracellular vesicles can be obtained by centrifuging milk, preferably by first centrifuging the milk at 1,600 to 2,400 g for 5 to 15 minutes and then at 7,000 to 13,000 g for 30 to 50 minutes, The supernatant obtained by the first centrifugation is centrifuged at 30,000 to 40,000 g for 40 to 80 minutes, and the supernatant obtained by the second centrifugation is centrifuged at 50,000 to 150,000 g for 40 to 80 minutes. It can be obtained by
상기 항암제는 옥살리플라틴 일 수 있다.The anticancer agent may be oxaliplatin.
상기 암 질환은 결장암, 직장암 및 유방암으로 이루어진 군에서 선택된 어느 하나 일 수 있으며, 이에 한정되는 것은 아니다.The cancer disease may be any one selected from the group consisting of colon cancer, rectal cancer and breast cancer, but is not limited thereto.
상기 우유 세포외소포체는 평균입경이 50 내지 500 nm일 수 있다.The milk extracellular vesicles may have an average particle diameter of 50 to 500 nm.
상기 우유 세포외소포체는 암 세포 생존 억제 및 암 세포자멸사를 유도시킬 수 있다.The milk extracellular vesicles can inhibit cancer cell survival and induce cancer apoptosis.
본 발명의 다른 구체예에서, 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다. In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, One or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants may be further included.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 일실시예에 따르면, 상기 약학 조성물은 정맥내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다. According to one embodiment of the present invention, the pharmaceutical composition is administered through intravenous, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. It can be administered to a subject in a manner.
본 발명에 따른 유효성분의 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있고, 1일 투여량이 0.01 mg/kg 내지 200 mg/kg, 바람직하게는 0.1 mg/kg 내지 200 mg/kg, 보다 바람직하게는 0.1 mg/kg 내지 100 mg/kg 일 수 있다. 투여는 하루에 한번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다. The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug type, the route and duration of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg. /kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, and more preferably 0.1 mg/kg to 100 mg/kg. Administration may be administered once a day or divided into several times, and the scope of the present invention is not limited thereby.
또한, 본 발명은 GE11 펩티드가 표지되고, 항암제가 로딩된 우유 세포외소포체를 포함하는 암 질환 예방 또는 개선용 건강식품용 조성물을 제공한다.In addition, the present invention provides a health food composition for preventing or improving cancer disease, comprising milk extracellular vesicles labeled with the GE11 peptide and loaded with an anticancer agent.
상기 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다.The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.
그밖에 천연 과일 주스, 합성 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품 조성물은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알코올 및 비타민 복합제 중 어느 하나의 형태일 수 있다.In addition, it may contain fruit flesh for the production of natural fruit juice, synthetic fruit juice and vegetable beverages. These components may be used independently or in combination. In addition, the health functional food composition is any one form of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex can be
또한, 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합 여부는 다른 규정이 없는 한 식품의약품안전청에 승인된 식품첨가물공전의 총칙 및 일반 시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.In addition, the health functional food may additionally contain food additives, and the suitability as a "food additive" is determined according to the general rules of the Food Additive Code and general test methods approved by the Korea Food and Drug Administration unless otherwise specified. It is judged according to the relevant standards and standards.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류 첨가 알칼리제, 보존료제제, 타르색소 제제 등의 혼합 제제류 등을 들 수 있다.Examples of items listed in the “Food Additives Codex” include, for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goreng pigment, guar gum, L -Mixed preparations such as sodium glutamate preparations, noodle-added alkali preparations, preservative preparations, tar color preparations, and the like.
이때, 건강기능식품을 제조하는 과정에서 식품에 첨가되는 유효성분은 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100 중량부에 1 중량부 내지 90 중량부 포함되도록 첨가될 수 있다.At this time, the content of the active ingredient added to the food in the process of manufacturing the health functional food may be appropriately increased or decreased as necessary, and preferably may be added so that 1 part by weight to 90 parts by weight is included in 100 parts by weight of the food. .
또한, 본 발명은 a) 우유를 원심분리하여 우유 유래 세포외소포체를 분리하는 단계; b) 상기 a) 단계에서 수득된 우유 유래 세포외소포체를 콜레스테롤-폴리에틸렌글리콜(PEG)-DBCO(Dibenzocyclooctyne)에 혼합한 후, GE11 펩티드에 혼합하여 GE11 표지를 부착하는 단계; 및 c) 상기 b) 단계에서 수득된 GE11 표지 우유 유래 세포외소포체를 항암약물과 인큐베이션하는 단계;를 포함하는 상기 약학 조성물 제조방법을 제공한다.In addition, the present invention a) separating the milk-derived extracellular vesicles by centrifuging the milk; b) mixing the milk-derived extracellular vesicles obtained in step a) with cholesterol-polyethylene glycol (PEG)-DBCO (Dibenzocyclooctyne), and then mixing with GE11 peptide to attach a GE11 label; and c) incubating the GE11-labeled milk-derived extracellular vesicles obtained in step b) with an anticancer drug.
상기 a) 단계에서 원심분리는 우유를 1,600 내지 2,400 g에서 5 내지 15분 및 그 후에 7,000 내지 13,000 g에서 30 내지 50분 동안 1차 원심분리하고, 상기 1차 원심분리로 수득한 상층액을 30,000 내지 40,000 g에서 40 내지 80분간 2차 원심분리하며, 상기 2차 원심분리로 수득한 상층액을 50,000 내지 150,000 g에서 40 내지 80분 동안 3차 원심분리하는 것일 수 있다.In step a), the centrifugation is performed by first centrifuging the milk at 1,600 to 2,400 g for 5 to 15 minutes and then at 7,000 to 13,000 g for 30 to 50 minutes, and the supernatant obtained by the first centrifugation is 30,000 Second centrifugation at 40,000 g for 40 to 80 minutes, and the supernatant obtained by the second centrifugation may be third centrifugation at 50,000 to 150,000 g for 40 to 80 minutes.
상기 b) 단계에서 GE11 펩티드는 아자이드가 개질된 (azide-modified) 것일 수 있다.In step b), the GE11 peptide may be azide-modified.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 다만 하기의 실시예 등은 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예 등에 한정되는 것은 아니다. 본 발명의 실시예 등은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention and the like are provided to more completely explain the present invention to those skilled in the art.
[[ 실험예Experimental example 1] One] 우유 유래milk origin 세포외소포체를extracellular vesicles 고온 및 낮은 pH 처리 후, After high temperature and low pH treatment, 세포외소포체extracellular endoplasmic reticulum 마커 CD9의 발현 분석 Expression analysis of the marker CD9
우유 유래 세포외소포체는 밀도 구배 초원심분리를 이용하여 분리하였다. 흔히 구할 수 있는 상업용 저온 살균 우유를 2,000 g에서 10분 동안, 10,000 g에서 40분 동안 원심분리 한 후, 상층액을 모아 추가로 1시간 동안 35,000 g에서 원심분리하였다. 그 후, 추가로 1시간 동안 100,000 g에서 밀도 구배 초원심분리를 하여 우유 유래 세포외소포체를 정제하였다. 밀도 구배는 10 % 및 50 % 요오딕사놀 (iodixanol; Sigma Aldrich, MO, USA)을 사용하여 진행하였는데, 50 % 요오딕사놀을 초원심분리기 튜브의 아래쪽에 넣은 후, 섞이지 않게 10 % 요오딕사놀 및 우유 샘플을 추가하였다. 상기 조건에 따라 우유 유래 세포외소포체는 10 % 및 50 % 요오딕사놀의 사이에서 얻었다. Milk-derived extracellular vesicles were isolated using density gradient ultracentrifugation. Commonly available commercial pasteurized milk was centrifuged at 2,000 g for 10 minutes and 10,000 g for 40 minutes, then the supernatant was collected and centrifuged at 35,000 g for an additional hour. Thereafter, milk-derived extracellular vesicles were purified by density gradient ultracentrifugation at 100,000 g for an additional 1 hour. The density gradient was carried out using 10% and 50% iodixanol (Sigma Aldrich, MO, USA). After putting 50% iodixanol at the bottom of the ultracentrifuge tube, 10% iodixanol was added without mixing. and milk samples were added. According to the above conditions, milk-derived extracellular vesicles were obtained between 10% and 50% iodixanol.
온도에 따른 우유 유래 세포외소포체의 변화를 확인하기 위하여, 추출된 세포외소포체는 3시간 동안 고온 (75 ℃) 처리했고, pH에 대한 변화를 알아보기 위하여 추출된 세포외소포체는 중성 (pH 7.2) 또는 낮은 pH (pH 5) 조건에 노출하였다. 우유 유래 세포외소포체의 총 단백질 농도는 Bio-Rad 단백질 분석 키트 (Hercules, CA, USA)를 사용하여 Bradford 분석을 통하여 확인하였다. 모든 실험은 n=3으로 진행했다.In order to confirm the change of milk-derived extracellular vesicles according to temperature, the extracted extracellular vesicles were treated at high temperature (75 ℃) for 3 hours, and the extracted extracellular vesicles were neutral (pH 7.2) to determine the change in pH. ) or low pH (pH 5) conditions. The total protein concentration of milk-derived extracellular vesicles was confirmed by Bradford assay using a Bio-Rad protein assay kit (Hercules, CA, USA). All experiments were conducted with n = 3.
그 후, 웨스턴 블롯 분석을 시행하였다. 우유 유래 세포외소포체의 단백질 추출물은 10 내지 15 %의 SDS-PAGE를 통해 분리된 다음 0.2 μm PVDF 막으로 옮겼다. 차단 스텝은 3 % 탈지유 또는 무단백질 차단 완충액 (Thermo Fishers)을 사용하여 1시간 동안 수행하고 CD9에 대한 1차 항체 (1:1000, NB500-494, Novus Biologicals)를 실온에서 2시간 동안 반응시켰다. 0.05 % TBS-T로 세 번 세척한 후, PVDF 막을 HRP-접합된 염소 항-마우스 IgG 2차 항체 (Santa Cruz)와 함께 인큐베이션하였다. 밴드는 향상된 화학발광 (ECL; Amersham Pharmacia Biotech, England, UK)을 이용하여 검출했다.Then, Western blot analysis was performed. Protein extracts from milk-derived extracellular vesicles were separated by 10 to 15% SDS-PAGE and then transferred to a 0.2 μm PVDF membrane. The blocking step was performed for 1 hour using 3% skim milk or protein-free blocking buffer (Thermo Fishers) and reacted with a primary antibody (1:1000, NB500-494, Novus Biologicals) at room temperature for 2 hours. After washing three times with 0.05% TBS-T, the PVDF membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz). Bands were detected using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, England, UK).
그 결과, 도 1A, 1B 그리고 1C에 따를 때, 우유 유래 세포외소포체는 293T 세포에서 유래한 세포외소포체에 비하여 높은 온도에서도 세포외소포체 마커 단백질 CD9가 발현되는 것을 확인하였다. 또한, 도 1D, 1E 그리고 1F에 따를 때, 우유에서 유래한 세포외소포체는 293T 세포에서 유래한 세포외소포체에 비하여 낮은 pH에서도 세포외소포체 마커 단백질 CD9가 발현되는 것을 확인하였다.As a result, according to Figures 1A, 1B and 1C, it was confirmed that the milk-derived extracellular vesicles expressed the extracellular vesicle marker protein CD9 even at a higher temperature than the extracellular vesicles derived from 293T cells. In addition, according to Figures 1D, 1E and 1F, it was confirmed that the extracellular vesicles derived from milk expressed the extracellular vesicles marker protein CD9 even at low pH compared to the extracellular vesicles derived from 293T cells.
[[ 실험예Experimental example 2] 콜레스테롤- 2] Cholesterol- 폴리에틸렌글리콜polyethylene glycol (PEG)-비오틴(biotin)을 사용하여 우유 Milk using (PEG)-biotin 세포외소포체에in the extracellular endoplasmic reticulum 콜레스테롤- cholesterol- 폴리에틸렌글리콜polyethylene glycol (PEG)-(PEG)- DBCO(Dibenzocyclooctyne)의of dibenzocyclooctyne (DBCO). 표지 정도를 확인 Check the degree of cover
도트 분석을 통하여 우유 유래 세포외소포체에 콜레스테롤-PEG-비오틴 결합을 분석하였다. 우유 유래 세포외소포체; 콜레스테롤-PEG-비오틴; 및 우유 세포외소포체 및 콜레스테롤-PEG-비오틴 혼합물;을 요오딕사놀 밀도 구배 초원심분리를 통하여 분리한 후, 분리된 용액들을 회수했다. 상기 분리된 용액들은 0.2 μm 니트로셀룰로오스막 (Amersham, Westborough, MA, USA)에 로딩한 후, 멤브레인을 50 ℃ 인큐베이터에서 밤새 배양하여 건조시키고, 막을 3 % 탈지유 또는 무단백질 차단 완충액 (Thermo Fishers)으로 1시간 동안 차단하고, CD9 (1:2000, NB500-494, Novus Biologicals) 및 비오틴 (1:2000, Thermo 피셔)를 실온에서 2시간 동안 인큐베이션했다. 그 후, 0.05 % TBS-T로 세 번 세척한 후, 막을 HRP-접합된 염소 항-마우스 IgG 2차 항체 (Santa Cruz)와 함께 반응시켰다. 밴드는 향상된 화학발광 (ECL; Amersham Pharmacia Biotech, England, UK)을 이용하여 검출했다. 그리고 위와 같은 방법을 이용하여 우유 유래 세포외소포체 및 콜레스테롤-PEG-비오틴을 총 6개의 다양한 배율 (1) 1:0.02, 2) 1:0.04, 3) 1:0.08, 4) 1:0.17, 5) 1:0.33 및 6) 1:0.67)로 혼합하여 반응 정도를 확인하였다.Cholesterol-PEG-biotin binding to milk-derived extracellular vesicles was analyzed by dot analysis. Milk-derived extracellular vesicles; cholesterol-PEG-biotin; and milk extracellular vesicles and cholesterol-PEG-biotin mixture; after separation through iodixanol density gradient ultracentrifugation, the separated solutions were recovered. The separated solutions were loaded on a 0.2 μm nitrocellulose membrane (Amersham, Westborough, MA, USA), the membrane was incubated overnight in an incubator at 50 ° C and dried, and the membrane was mixed with 3% skim milk or protein-free blocking buffer (Thermo Fishers). Blocked for 1 hour, and incubated with CD9 (1:2000, NB500-494, Novus Biologicals) and biotin (1:2000, Thermo Fisher) for 2 hours at room temperature. Then, after washing three times with 0.05% TBS-T, the membrane was reacted with HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz). Bands were detected using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, England, UK). And using the above method, milk-derived extracellular vesicles and cholesterol-PEG-biotin were prepared at a total of six different magnifications (1) 1:0.02, 2) 1:0.04, 3) 1:0.08, 4) 1:0.17, 5 ) 1:0.33 and 6) 1:0.67) to confirm the degree of reaction.
그 결과, 도 1A에 따를 때, 우유 유래 세포외소포체는 콜레스테롤-PEG-비오틴으로 표지되어 F8층에서 검출됨을 확인하였다. 또한, 도 1B 및 1C에 의할 때, 우유 유래 세포외소포체 및 콜레스테롤-PEG-비오틴은 농도에 비례하여 표지 정도가 증가하지만, 1:0.33의 농도배율일 때 가장 효율적으로 반응하였고 그 이상에선 과포화 상태인 것을 확인하였다.As a result, according to Figure 1A, it was confirmed that milk-derived extracellular vesicles were labeled with cholesterol-PEG-biotin and detected in the F8 layer. In addition, according to Figures 1B and 1C, milk-derived extracellular vesicles and cholesterol-PEG-biotin increase the degree of labeling in proportion to the concentration, but reacted most efficiently at a concentration ratio of 1:0.33 and supersaturated above that. status was confirmed.
[[ 실험예Experimental example 3] GE11 펩티드가 표지된 3] GE11 peptide labeled 우유 유래milk origin 세포외소포체에in the extracellular endoplasmic reticulum 옥살리플라틴oxaliplatin (oxaliplatin)을 로딩한 후 세포외소포체 특성 분석 Analysis of extracellular vesicle characteristics after loading (oxaliplatin)
우유 유래 세포외소포체에 GE 펩타이드를 표지했다. 900 μg의 우유 유래 세포외소포체를 300 μg의 콜레스테롤-폴리에틸렌글리콜(PEG)-DBCO(Dibenzocyclooctyne) (이하, 콜레스테롤-PEG-DBCO라함)와 혼합하고 실온에서 1시간 동안 인큐베이션하였다. DBCO가 포함된 우유 유래 세포외소포체를 10 내지 50 % 요오딕사놀을 이용하여 밀도 구배 초원심분리 후 추출하였다. 그런 다음 DBCO가 포함된 우유 유래 세포외소포체를 200 μg 아자이드가 개질된 (azide-modified GE11) 펩티드와 혼합하고 실온에서 1시간 동안 반응시켰다. GE11 펩티드가 표지된 우유 유래 세포외소포체는 10 내지 50 % 요오딕사놀를 이용하여 구배 초원심분리 후 추출하였다. 대조군 우유 유래 세포외소포체는 콜레스테롤-PEG-DBCO 및 GE11 펩티드 없이 동일하게 진행하였다. 콜레스테롤-PEG-DBCO는 Nanocs (NY, USA)에서 구입하여 사용했으며, GE11 펩티드 및 MYC-GE11 펩타이드는 펩트론사(대전, 대한민국)에서 합성하였다. Milk-derived extracellular vesicles were labeled with GE peptides. 900 μg of milk-derived extracellular vesicles were mixed with 300 μg of cholesterol-polyethylene glycol (PEG)-dibenzocyclooctyne (DBCO) (hereinafter referred to as cholesterol-PEG-DBCO) and incubated at room temperature for 1 hour. Milk-derived extracellular vesicles containing DBCO were extracted after density gradient ultracentrifugation using 10 to 50% iodixanol. Then, the milk-derived extracellular vesicles containing DBCO were mixed with 200 μg of azide-modified GE11 peptide and reacted at room temperature for 1 hour. Milk-derived extracellular vesicles labeled with the GE11 peptide were extracted after gradient ultracentrifugation using 10 to 50% iodixanol. Control milk-derived extracellular vesicles were processed in the same way without cholesterol-PEG-DBCO and GE11 peptides. Cholesterol-PEG-DBCO was purchased and used from Nanocs (NY, USA), and GE11 peptide and MYC-GE11 peptide were synthesized from Peptron (Daejeon, Korea).
이렇게 생성된 GE11 펩티드가 표지된 우유 유래 세포외소포체에 항암 약물인 옥살리플라틴을 로딩하였다. 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 1000 μg/mL의 옥살리플라틴과 혼합하고, 40 ℃에서 12시간 동안 반응시켰다. 그런 다음 10 내지 50 % 요오딕사놀을 이용하여 밀도 구배 초원심분리를 하였고, 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 추출하였고, 전자현미경, 나노입자추적분석법 (nanoparticle tracking analysis; 이하, NTA라함) 및 웨스턴 블롯을 통하여 특성이 분석하였다.The resulting GE11 peptide-labeled milk-derived extracellular vesicles were loaded with oxaliplatin, an anticancer drug. Milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles were mixed with 1000 μg/mL of oxaliplatin and reacted at 40° C. for 12 hours. Then, density gradient ultracentrifugation was performed using 10 to 50% iodixanol, and oxaliplatin-loaded milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles were extracted, and electron microscopy and nanoparticle tracking were performed. Characteristics were analyzed through nanoparticle tracking analysis (hereinafter referred to as NTA) and Western blot.
세포외소포체들의 입자 농도 및 크기는 NTA를 통하여 분석하였으며, 688 nm 레이저가 장착된 NanoSight LM10-HS 시스템 (Malvern Instruments Ltd.)을 사용하고, 그 결과는 NTA 소프트웨어 버전 2.3 (Malvern Instruments Ltd.)을 이용하여 분석하였다.The particle concentration and size of extracellular vesicles were analyzed through NTA, using a NanoSight LM10-HS system (Malvern Instruments Ltd.) equipped with a 688 nm laser, and the results were analyzed using NTA software version 2.3 (Malvern Instruments Ltd.). analyzed using
세포외소포체 마커를 확인하기 위하여 웨스턴 블롯 분석을 시행하였다. 우유 유래 세포외소포체에서 추출된 단백질은 10 내지 15 %의 SDS-PAGE를 통해 분리된 다음 0.2μm PVDF 막에 옮겼다. 차단은 3 % 탈지유 또는 무단백질 차단 완충액 (Thermo Fishers)을 1시간 동안 반응시켰으며, 1차 항체로 CD81 (1:1000, SC-166029, Santa Cruz) 및 CD9 (1:1000, NB500-494, Novus Biologicals)을 사용하여 실온에서 2시간 동안 반응시켰다. 그 후, 0.05 % TBS-T로 세 번 세척한 후, 막을 HRP-접합된 염소 항-마우스 IgG 2차 항체 (Santa Cruz)와 함께 반응시켰다. 밴드는 향상된 화학발광 (ECL; Amersham Pharmacia Biotech, England, UK)을 이용하여 검출했다.Western blot analysis was performed to confirm the extracellular endoplasmic reticulum markers. Proteins extracted from milk-derived extracellular vesicles were separated by 10 to 15% SDS-PAGE and then transferred to a 0.2 μm PVDF membrane. Blocking was performed with 3% skim milk or protein-free blocking buffer (Thermo Fishers) for 1 hour, and CD81 (1:1000, SC-166029, Santa Cruz) and CD9 (1:1000, NB500-494, Novus Biologicals) was used at room temperature for 2 hours. Then, after washing three times with 0.05% TBS-T, the membrane was reacted with HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz). Bands were detected using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, England, UK).
그 결과, 도 3A에 따를 때, 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체는 옥살리플라틴의 로딩 이후에 GE11의 표지 여부에 상관 없이 일정한 세포소포체의 외형을 가지고 있음을 확인했다. 또한, 도 3B 및 C에 따를 때, 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체는 옥살리플라틴의 로딩 이후에 GE11의 표지와 상관 없이 세포외소포체의 크기는 50 내지 500 nm 정도이며, 평균은 옥살리플라틴 로딩 우유 유래 세포외 소포체는 207.24 nm 그리고 GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외소포체는 217.71 nm 로 균일한 크기로 존재하는 것을 확인했다. 또한, 도 3D에 따를 때, 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체는 세포외소포체 마커인 CD9 및 CD81이 발현되는 것을 확인하였다.As a result, according to FIG. 3A, it was confirmed that milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles had a constant appearance of endoplasmic reticulum regardless of whether or not GE11 was labeled after oxaliplatin loading. 3B and C, the milk-derived extracellular vesicles and the milk-derived extracellular vesicles labeled with the GE11 peptide have a size of 50 to 500 nm regardless of the GE11 label after loading with oxaliplatin. , It was confirmed that the average size of the oxaliplatin-loaded milk-derived extracellular vesicles was 207.24 nm and the GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular vesicles were 217.71 nm in uniform size. In addition, according to Figure 3D, it was confirmed that the milk-derived extracellular vesicles loaded with oxaliplatin and the milk-derived extracellular vesicles labeled with the GE11 peptide expressed the extracellular vesicle markers CD9 and CD81.
[[ 실험예Experimental example 4] GE11 4] GE11 펩트드가Peptide 표지된 labeled 우유 유래milk origin 세포외소포체의extracellular endoplasmic reticulum 옥살리플라틴oxaliplatin 로딩 후 특성 분석 Characterization after loading
GE11 펩티드가 표지된 우유 유래 세포외소포체에 GE11 펩트드가 표지 되었는지 확인하기 위하여 웨스턴 블롯 분석을 시행하였다. 우유 유래 세포외소포체에서 추출된 단백질은 10 내지 15 %의 SDS-PAGE를 통해 분리된 다음 0.2 μm PVDF 막에 옮겼다. 차단은 3 % 탈지유 또는 무단백질 차단 완충액 (Thermo Fishers)을 1시간 동안 반응시켰으며, 1차 항체로 GE11 펩티드를 사용하여 실온에서 2시간 동안 반응시켰다. 그 후, 0.05 % TBS-T로 세 번 세척한 후, 막을 HRP-접합된 염소 항-마우스 IgG 2차 항체 (Santa Cruz)와 함께 반응시켰다. 밴드는 향상된 화학발광 (ECL; Amersham Pharmacia Biotech, England, UK)을 이용하여 검출했다.Western blot analysis was performed to confirm whether the GE11 peptide was labeled on the milk-derived extracellular vesicles labeled with the GE11 peptide. Proteins extracted from milk-derived extracellular vesicles were separated by 10 to 15% SDS-PAGE and then transferred to a 0.2 μm PVDF membrane. For blocking, 3% skim milk or protein-free blocking buffer (Thermo Fishers) was reacted for 1 hour, and GE11 peptide was used as the primary antibody for 2 hours at room temperature. Then, after washing three times with 0.05% TBS-T, the membrane was reacted with HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz). Bands were detected using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, England, UK).
옥살리플라틴의 정량 분석을 위해서는 서울대학교 국립대학간연구시설센터 (NCIRF)에서 총 50 μg의 우유 유래 세포외소포체들을 이용하여 ICP-MS로 백금 분석을 시행했다. 옥살리플라틴 방출 역학을 분석하기 위하여 옥살리플라틴이 로딩된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체를 0.1 mL의 PBS로 현탁하고 Slide-A-Lyzer MINI 투석 장치 (10kDa MWCO)에 로딩하고 37 ℃에서 1 mL의 PBS에 대해 투석했다. 그 후, 상층액을 0, 2, 4, 6, 12, 24 및 36시간 후에 각각 회수했다. 옥살리플라틴의 농도는 위에 언급했듯이 ICP-MS로 정량하였다. 모든 실험은 n=3으로 시행되었다.For quantitative analysis of oxaliplatin, platinum analysis was performed by ICP-MS using a total of 50 μg of milk-derived extracellular vesicles at Seoul National University National Intercollegiate Research Facility Center (NCIRF). To analyze oxaliplatin release kinetics, oxaliplatin-loaded milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles were suspended in 0.1 mL of PBS and loaded into a Slide-A-Lyzer MINI dialysis device (10 kDa MWCO). Dialyzed against 1 mL of PBS at 37 °C. Thereafter, the supernatant was recovered after 0, 2, 4, 6, 12, 24 and 36 hours, respectively. The concentration of oxaliplatin was quantified by ICP-MS as mentioned above. All experiments were conducted with n=3.
그 결과, 도 4A에 따를 때, 옥살리플라틴이 로딩된 우유 유래 세포외소포체 대비 옥살리플라틴이 로딩된 GE11 팹티드가 표지된 우유 유래 세포외소포체에만 GE11이 발현되는 것을 확인하였다. 또한, 도 4B에 따를 때, 옥살리플라틴은 GE11의 표지와 상관 없이 우유 유래 세포외소포체들에서 옥살리플라틴 약물 농도의 증가에 따라 로딩량이 증가함을 확인하였다. 또한, 도 4C에 따를 때, 옥살리플라틴의 우유 유래 세포외소포체에서 방출량은 GE11의 표지와 상관없는 것을 확인하였다.As a result, according to FIG. 4A , it was confirmed that GE11 was expressed only in milk-derived extracellular vesicles labeled with oxaliplatin-loaded GE11 peptid compared to oxaliplatin-loaded milk-derived extracellular vesicles. In addition, according to FIG. 4B, it was confirmed that the loading amount of oxaliplatin increased with the increase of the oxaliplatin drug concentration in milk-derived extracellular vesicles regardless of the label of GE11. In addition, according to Figure 4C, it was confirmed that the amount of oxaliplatin released from milk-derived extracellular vesicles was not related to the label of GE11.
[[ 실험예Experimental example 5]. GE11 펩티드가 표지된 5]. GE11 peptide labeled 우유 유래milk origin 세포외소포체의extracellular endoplasmic reticulum 암세포 표적 효과 확인 Confirmation of cancer cell targeting effect
인간 결장암 세포주 (이하, SNU-C5라함) 및 인간 유방암 세포주 (이하, MDA-MB-231이라함)는 10 %(v/v) 소태아혈청, 100 U/mL 페니실린 및 스트렙토마이신 (Gibco BRL, Gaithersburg, MD, USA)이 첨가된 Roswell Park Memorial Institute 1640 배지로 배양되었다. 인간 제대 정맥 내피 세포 (HUVEC)(Thermo Fisher Scientific)를 저혈청 성장 보충제 (LSGS)(Thermo Fisher Scientific)가 보충된 Medium 200에서 배양했다. 그리고 모든 세포는 37 ℃의 가습된 5 % 이산화탄소 (CO2) 인큐베이터에서 동일한 조건으로 배양했다. 그 후, 세포들의 EGFR (Epidermal Growth Factor Receptor) 마커를 확인하기 위하여 웨스턴 블롯 분석을 시행하였다. 세포들에게서 추출된 단백질은 10 내지 15 %의 SDS-PAGE를 통해 분리된 다음 0.2 μm PVDF 막에 옮겼다. 차단은 3 % 탈지유 또는 무단백질 차단 완충액 (Thermo Fishers)을 1시간 동안 반응시켰으며, 1차 항체로 EGFR (1:1000) 그리고 β-actin (1:1000)을 사용하여 실온에서 2시간 동안 반응시켰다. 그 후 0.05 % TBS-T로 세 번 세척한 후, 막을 HRP-접합된 염소 항-마우스 IgG 2차 항체 (Santa Cruz)와 함께 반응시켰다. 밴드는 향상된 화학발광 (ECL; Amersham Pharmacia Biotech, England, UK)을 이용하여 검출했다.Human colon cancer cell line (hereinafter referred to as SNU-C5) and human breast cancer cell line (hereinafter referred to as MDA-MB-231) were prepared using 10% (v/v) fetal bovine serum, 100 U/mL penicillin and streptomycin (Gibco BRL, Gaithersburg, MD, USA) supplemented with Roswell Park Memorial Institute 1640 medium. Human umbilical vein endothelial cells (HUVEC) (Thermo Fisher Scientific) were cultured in
우유 유래 세포외소포체에 형광 표지를 하기 위해 세포외소포체를 5 μg/mL의 CellMask™ 오렌지색 원형질막 얼룩 (Sigma Aldrich)으로 처리하고 실온에서 1시간 동안 인큐베이션했다. 그 후, 밀도 구배 초원심분리를 사용하여 CMO로 표지된 우유 유래 세포외소포체를 분리했다. SNU-C5 및 MDA-MB-231 세포를 37 ℃ 인큐베이터에서 2시간 동안 CMO 표지된 우유 세포외소포체와 반응시키고, 유세포 분석기 (Sysmex Partec) 및 공초점 현미경 (Leica, Wetzlar, Hesse, Germany)을 사용하여 암 세포내 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체의 흡수를 분석했다.To fluorescently label milk-derived extracellular vesicles, the extracellular vesicles were treated with 5 μg/mL CellMask™ orange plasma membrane stain (Sigma Aldrich) and incubated for 1 hour at room temperature. Thereafter, milk-derived extracellular vesicles labeled with CMO were isolated using density gradient ultracentrifugation. SNU-C5 and MDA-MB-231 cells were reacted with CMO-labeled milk extracellular vesicles for 2 hours in a 37 °C incubator, using flow cytometry (Sysmex Partec) and confocal microscopy (Leica, Wetzlar, Hesse, Germany). Uptake of milk-derived extracellular vesicles and milk-derived extracellular vesicles labeled with the GE11 peptide was analyzed in cancer cells.
그 결과, 도 5A에 따를 때, 암 마커인 EGFR은 인간 제대 정맥 내피 세포 (HUVEC)에 발현되지 않는 반면에 SNU-C5 및 MDA-MB-231에서는 EGFR이 발현된 것을 확인하였다. 또한, 도 5B에 따를 때, GE11 펩티드가 표지된 우유 유래 세포외소포체는 대조군 우유 유래 세포외소포체에 비하여 SNU-C5 및 MDA-MB-231을 표적하여 각 45.9 % 및 40.6 % 반응한 것을 확인하였다. 또한, 도 5C 및 D에 따를 때, CMO로 염색된 GE11가 표지된 우유 유래 세포외소포체는 대조군 우유 유래 세포외소포체에 비하여 SNU-C5 및 MDA-MB-231을 표적하여 반응하였으며, 암 세포 및 CMO 형광의 위치가 동일하다는 것을 확인하였다.As a result, according to FIG. 5A, it was confirmed that EGFR, a cancer marker, was not expressed in human umbilical vein endothelial cells (HUVEC), whereas EGFR was expressed in SNU-C5 and MDA-MB-231. In addition, according to FIG. 5B, it was confirmed that the milk-derived extracellular vesicles labeled with the GE11 peptide reacted 45.9% and 40.6%, respectively, to target SNU-C5 and MDA-MB-231 compared to the control milk-derived extracellular vesicles. . In addition, according to Figure 5C and D, milk-derived extracellular vesicles stained with CMO and labeled with GE11 reacted by targeting SNU-C5 and MDA-MB-231 compared to control milk-derived extracellular vesicles, cancer cells and It was confirmed that the position of the CMO fluorescence was the same.
[[ 실험예Experimental example 6] GE11 펩티드가 표지된 6] GE11 peptide labeled 옥살리플라틴oxaliplatin 로딩 loading 우유 유래milk origin 세포외소포체의extracellular endoplasmic reticulum 암세포 생존률 저하 및 세포사멸사 유도 효과 확인 Confirmation of cancer cell viability reduction and apoptosis induction effect
SNU-C5 및 MDA-MB-231을 96-웰 배양 플레이트 (5,000개 세포/웰)에 24시간 동안 인큐베이션하였다. 그 후, 세포들을 우유 유래 세포외소포체, GE11 펩티드가 표지된 우유 유래 세포외소포체, 옥살리플라틴 로딩 우유 유래 세포외소포체, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외소포체 및 옥살리플라틴을 처리했다 (모든 샘플은 옥살리플라틴 5μM이 처리되었고, 우유 유래 세포외소포체들은 동일한 양이 처리되었다). 이후 EZ-Cytox (DoGenBio, Seoul, Republic of Korea)를 이용하여 세포독성 분석을 수행하였고, 수행 방법은 제조사에서 제안해준 방법을 따라 시행되었다. 450 nm에서의 흡광도는 마이크로플레이트 리더 (BMG Labtech, Ortenberg, Germany)로 측정하였다.SNU-C5 and MDA-MB-231 were incubated in 96-well culture plates (5,000 cells/well) for 24 hours. Then, the cells were treated with milk-derived extracellular vesicles, GE11 peptide-labeled milk-derived extracellular vesicles, oxaliplatin-loaded milk-derived extracellular vesicles, GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular vesicles, and oxaliplatin (all samples was treated with 5 μM of oxaliplatin, and the same amount of milk-derived extracellular vesicles). Subsequently, cytotoxicity analysis was performed using EZ-Cytox (DoGenBio, Seoul, Republic of Korea), and the method was performed according to the method suggested by the manufacturer. Absorbance at 450 nm was measured with a microplate reader (BMG Labtech, Ortenberg, Germany).
세포자멸사 분석을 위하여 유세포분석을 이용하였다. SNU-C5 및 MDA-MB-231에서 세포 사멸을 확인하기 위하여 세포는 FITC (annexin V-fluorescein isothiocyanate) 및 PI (propidium iodide)(Sigma Aldrich)로 염색되었다. 형광 강도는 CyFlow Cube 8 (Sysmex Partec, M
그 결과, 도 6A 및 6B에 따를 때, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외소포체는 다른 대조군들에 비하여 SNU-C5 및 MDA-MB-231의 생존율을 감소시키는 것을 확인하였다. 또한, 도 6C , 6D 및 6E에 따를 때, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외소포체는 다른 대조군들에 비하여 SNU-C5 및 MDA-MB-231의 세포 자멸사를 유도시키는 것을 확인하였다.As a result, according to FIGS. 6A and 6B, it was confirmed that the GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular vesicles reduced the viability of SNU-C5 and MDA-MB-231 compared to other control groups. In addition, according to Figures 6C, 6D and 6E, it was confirmed that the GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular vesicles induced apoptosis of SNU-C5 and MDA-MB-231 compared to other control groups.
[[ 실험예Experimental example 7] 종양이식 대장암 종양 동물 모델에서 7] Tumor transplantation in colorectal cancer tumor animal models 옥살리플라틴의of oxaliplatin 농도에 따른 종양 생성 저하 효과 확인 Confirmation of tumor formation reduction effect according to concentration
종양이식 대장암 종양 동물 모델을 만들기 위하여 SNU-C5 세포 (5 x 106 cells/head)를 수컷 BALB/c 누드 마우스의 오른쪽 옆구리에 피하 주사하였다. 종양의 부피가 50 mm3에 도달하면 GE11 펩티드가 표지된 우유 유래 세포외소포체 및 GE11 펩티드가 표지된 우유 유래 세포외소포체에 옥살리플라틴이 1, 2 및 5 mg/kg 로딩된 약물을 3일 또는 4일 간격으로 정맥 주사를 통해 4회 투여했다. 그 후, 조직 추출 날짜에 맞춰 조직을 채취하였으며, 종양의 크기를 측정하기 위하여 두 개의 수직 종양 치수 (a = 길이, b = 너비)를 캘리퍼스로 측정하고 부피 (V; mm3)를 공식 V = (a × b2)/2로 계산했다.Tumor transplantation SNU-C5 cells (5 x 10 6 cells/head) were subcutaneously injected into the right flank of male BALB/c nude mice to create an animal model for colorectal cancer tumors. When the tumor volume reached 50 mm 3 , GE11 peptide-labeled milk-derived extracellular vesicles and GE11 peptide-labeled milk-derived extracellular vesicles were loaded with oxaliplatin at 1, 2, or 5 mg/kg for 3 or 4 days. It was administered 4 times via intravenous injection at daily intervals. Then, the tissue was collected according to the tissue extraction date, and to measure the size of the tumor, two vertical tumor dimensions (a = length, b = width) were measured with calipers, and the volume (V; mm 3 ) was calculated using the formula V = It was calculated as (a × b 2 )/2.
그 결과, 도 7에 따를 때, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외포소체는 옥살리플라틴의 농도에 따라 종양 크기 억제의 유효성이 증가함을 확인하였다.As a result, according to FIG. 7 , it was confirmed that the GE11 peptide-labeled oxaliplatin-loaded milk-derived exosomes increased the effectiveness of tumor size suppression according to the concentration of oxaliplatin.
[실험예 8] 마우스 모델에서의 생체 내 항종양 효과[Experimental Example 8] In vivo antitumor effect in mouse model
종양이식 대장암 종양 동물 모델을 만들기 위하여 SNU-C5 세포 (5 x 106 cells/head)를 수컷 BALB/c 누드 마우스의 오른쪽 옆구리에 피하 주사하였다. 종양의 부피가 50 mm3에 도달하면 PBS, 옥살리플라틴 로딩 우유 유래 세포외소포체 (5 mg/kg), 옥살리플라틴 (5 mg/kg), 옥살리플라틴 (20 mg/kg) 및 GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외세포체 (5 mg/kg)을 3일 또는 4일 간격으로 정맥 주사를 통해 4회 투여 했다. 그 후, 조직 추출 날짜에 맞춰 조직을 채취하였고, 종양의 크기를 측정하기 위하여 두 개의 수직 종양 치수 (a = 길이, b = 너비)를 캘리퍼스로 측정하고 부피 (V; mm3)를 공식 V = (a × b2)/2로 계산했다.Tumor transplantation SNU-C5 cells (5 x 10 6 cells/head) were subcutaneously injected into the right flank of male BALB/c nude mice to create an animal model for colorectal cancer tumors. When tumor volume reaches 50 mm 3 , PBS, oxaliplatin-loaded milk-derived extracellular vesicles (5 mg/kg), oxaliplatin (5 mg/kg), oxaliplatin (20 mg/kg), and GE11 peptide-labeled oxaliplatin-loaded milk The derived extracellular cell body (5 mg/kg) was administered 4 times via intravenous injection every 3 or 4 days. Then, the tissue was collected according to the date of tissue extraction, and to measure the size of the tumor, two vertical tumor dimensions (a = length, b = width) were measured with calipers, and the volume (V; mm 3 ) was calculated using the formula V = It was calculated as (a × b 2 )/2.
그 결과, 도 8A 및 8B에 따를 때, GE11 펩티드가 표지된 옥살리플라틴 로딩 우유 유래 세포외소포체는 다른 대조군들에 비하여 대장 종양의 크기를 감소시키는 것을 확인하였다.As a result, according to FIGS. 8A and 8B , it was confirmed that the GE11 peptide-labeled oxaliplatin-loaded milk-derived extracellular vesicles reduced the size of colon tumors compared to other control groups.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.
Claims (11)
b) 상기 a) 단계에서 수득된 우유 유래 세포외소포체를 콜레스테롤-폴리에틸렌글리콜(PEG)-DBCO(Dibenzocyclooctyne)에 혼합한 후, GE11 펩티드에 혼합하여 GE11 표지를 부착하는 단계; 및
c) 상기 b) 단계에서 수득된 GE11 표지 우유 유래 세포외소포체를 항암약물과 인큐베이션하는 단계;를 포함하는 청구항 2의 약학 조성물 제조방법.a) separating milk-derived extracellular vesicles by centrifuging milk;
b) mixing the milk-derived extracellular vesicles obtained in step a) with cholesterol-polyethylene glycol (PEG)-DBCO (Dibenzocyclooctyne), and then mixing with GE11 peptide to attach a GE11 label; and
c) incubating the GE11-labeled milk-derived extracellular vesicles obtained in step b) with an anticancer drug;
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