KR20220051297A - Enhanced functional Phamaceutical composition of extracellular vesicles derived marine animal - Google Patents
Enhanced functional Phamaceutical composition of extracellular vesicles derived marine animal Download PDFInfo
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Abstract
Description
본 발명은 해양생물에서 유래된 세포외 기질에서 추출된 세포외 소포를 유효성분으로 하는 약학조성물에 대한 것이다. 보다 상세하게는 해양생물의 조직에서 세포외 기질을 추출하고 세포외 기질에 포함된 세포외 소포를 추출한 약학조성물로서 세포외 소포가 가진 기능이 그대로 발현되도록 처리된 약학조성물에 대한 것이다.The present invention relates to a pharmaceutical composition comprising, as an active ingredient, an extracellular vesicle extracted from an extracellular matrix derived from a marine organism. More specifically, it relates to a pharmaceutical composition obtained by extracting the extracellular matrix from the tissue of a marine organism and extracting the extracellular vesicles contained in the extracellular matrix, and treated to express the functions of the extracellular vesicles as they are.
기존의 연구를 통해 줄기세포는 미분화 세포로서 신체를 구성하는 여러 기관이나 조직을 구성하는 세포로 분화할 수 있음이 널리 알려져 있다. 또한, 줄기세포를 포함하는 여러 종류의 세포에서 유래된 세포외 기질은 유래된 세포가 지니고 있는 기능을 일정 부분 모사하는 것으로 알려져 있다. 이러한 기능의 모사는 세포외 기질에 포함된 여러 가지 성분에서 유래되는 것으로 알려져 있고, 세포외 기질에 포함된 세포외 소포로부터도 유래하는 것으로 알려져 있으나 정확한 기전은 밝혀지지 않은 상태이고 세포외 소포로부터 유래되는 모사에 관련된 정확한 기전에 대한 연구가 활발하게 진행되고 있다.Through existing research, it is widely known that stem cells are undifferentiated cells that can be differentiated into cells constituting various organs or tissues constituting the body. In addition, the extracellular matrix derived from various types of cells, including stem cells, is known to partially mimic the functions of the derived cells. It is known that the imitation of these functions is derived from various components contained in the extracellular matrix, and it is also known to originate from the extracellular vesicles contained in the extracellular matrix, but the exact mechanism is not known and is derived from extracellular vesicles. Research on the exact mechanism involved in the simulation is being actively conducted.
또한, 조직공학 및 재생의학 분야에서 세포 유래 세포외 기질(Extracellular matrix : ECM)을 이용하여 치료 또는 조직재생에 효과가 있는 약학조성물을 제조하거나 효과를 높이기 위한 시도가 활발하게 진행되고 있고, 현재까지 전 세계적으로 약 60여개의 이종 또는 동종 유래 세포외 기질을 이용한 의료기기가 FDA 허가를 받아 시판 중에 있다. 이러한 세포 유래 세포외 기질을 기반으로 한 의료기기는 조직 특이성을 가져 유래된 조직에 따라 생리적 특성이 서로 다르게 나타난다는 연구가 보고되고 있다.In addition, in the field of tissue engineering and regenerative medicine, attempts are actively being made to manufacture a pharmaceutical composition effective for treatment or tissue regeneration or to increase the effect by using a cell-derived extracellular matrix (ECM), and until now Around the world, about 60 medical devices using heterologous or allogeneic extracellular matrix have been approved by the FDA and are on the market. Research has been reported that medical devices based on such a cell-derived extracellular matrix have tissue specificity, and that physiological characteristics appear differently depending on the tissue from which they are derived.
하지만 이러한 세포외 기질을 활용하는 의료기기는 대부분 시트(sheet)의 형태나 스펀지 등의 제형에 한정되어 있어 외과적 시술이 필요한 경우가 많고 특히, 퇴행성 관절염의 경우 관절에 발생한 염증을 치료함과 동시에 연골조직을 보호하고 재생을 촉진하는 복합적인 치료가 필요하지만 연골세포 유래 세포외 기질의 경우 연골 조직을 보호하거나 연골 조직의 재생을 촉진하는 등 단일한 기능을 수행하는데 그쳐 치료가 완전하게 이루어지지 않거나 재발되는 경우가 많은 문제가 있다.However, most of the medical devices using the extracellular matrix are limited to the form of a sheet or a formulation such as a sponge, so surgical procedures are often required. Although a complex treatment that protects cartilage tissue and promotes regeneration is required, in the case of chondrocyte-derived extracellular matrix, it only performs a single function, such as protecting cartilage tissue or promoting the regeneration of cartilage tissue. There are many problems that recur.
또한, 세포외 소포의 추출도 종래의 세포 배양액을 이용하는 경우 세포를 배양하기 위한 시간 및 막대한 경제적 비용이 요구될 뿐만 아니라 실제 치료에 사용하기 위해 충분한 세포외 소포를 확보할 필요가 있으나 수율이 제한적인 문제점이 있다.In addition, extraction of extracellular vesicles also requires time and enormous economic cost for culturing cells when using a conventional cell culture medium, and it is necessary to secure sufficient extracellular vesicles for use in actual treatment, but the yield is limited. There is a problem.
본 발명은 상기와 같은 문제점을 해결하기 위한 것으로, 본 발명은 해양생물 유래 세포외 소포를 유효성분으로 하는 약학조성물을 제공한다.The present invention is to solve the above problems, and the present invention provides a pharmaceutical composition comprising an extracellular vesicle derived from a marine organism as an active ingredient.
본 발명은 해양생물에서 세포외 기질을 추출한 후 세포외 기질에서 추출된 세포외 소포를 포함하여 추출된 세포외 소포의 기능이 발현되는 세포외 소포 기능발현 약학조성물을 제공한다.The present invention provides an extracellular vesicle function expression pharmaceutical composition in which the function of the extracted extracellular vesicle is expressed, including the extracellular vesicle extracted from the extracellular matrix after extracting the extracellular matrix from the marine organism.
본 발명은 특히 퇴행성 관절염의 치료에 적용되어 염증 조절뿐만 아니라 조직 재생에도 효과가 있는 약학조성물을 제공한다.The present invention is particularly applied to the treatment of degenerative arthritis to provide a pharmaceutical composition that is effective not only in regulating inflammation but also in tissue regeneration.
상기와 같은 목적을 달성하기 위해 본 발명의 약학조성물은 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포를 유효성분으로 한다.In order to achieve the above object, the pharmaceutical composition of the present invention uses extracellular vesicles extracted from the extracellular matrix derived from marine organisms as an active ingredient.
또한, 상기 세포외 기질에서 추출한 세포외 소포는 해상 생물 중 해삼으로부터 추출된 것을 특징으로 한다.In addition, the extracellular vesicles extracted from the extracellular matrix are characterized in that extracted from sea cucumbers among marine organisms.
또한, 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 엑소좀인 것을 특징으로 한다.In addition, the extracellular vesicles extracted from the extracellular matrix derived from marine organisms are characterized in that they are exosomes.
또한, 상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 해양동물을 분쇄하여 세포외기질을 추출하고, 상기 추출된 세포외기질에 효소처리 단계를 거쳐 세포외 소포를 추출하는 것을 특징으로 한다.In addition, the extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are pulverized to extract the extracellular matrix, and the extracted extracellular matrix is subjected to an enzyme treatment step to extract the extracellular vesicles. do.
또한, 상기 세포외기질이 추출은 분쇄된 해양동물 조직을 교반하는 단계를 통해 해양동물 조직을 용해시키는 단계, 용해되지 않은 찌꺼기를 걸러주는 단계와 원심분리하는 단계를 통해 추출하는 것을 특징으로 한다.In addition, the extraction of the extracellular matrix is characterized in that the extraction is performed through a step of dissolving the marine animal tissue through agitation of the pulverized marine animal tissue, a step of filtering undissolved debris, and a step of centrifugation.
또한, 상기 추출된 세포외기질에 효소처리하는 단계는 추출된 세포외기질을 동결건조한 후 HCL과 펩신을 이용하여 세포외기질의 결합을 분해하는 단계, NaOH를 이용하여 중화시킨 후 투석하는 단계, 중화 투석된 세포외기질을 collagenase를 첨가한 후 교반하는 단계 및 원심 분리를 통해 찌꺼기를 제거하는 단계를 통해 세포외 소포를 추출하는 것을 특징으로 한다.In addition, the enzymatic treatment of the extracted extracellular matrix includes lyophilizing the extracted extracellular matrix and then dissolving the binding of the extracellular matrix using HCL and pepsin, neutralizing with NaOH and then dialysis, neutralization It is characterized in that the extracellular vesicles are extracted through the step of stirring the dialyzed extracellular matrix after adding collagenase and removing the debris through centrifugation.
또한, 상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포의 크기는 60~500nm 범위 내에 있는 것을 특징으로 한다.In addition, the size of the extracellular vesicles extracted from the extracellular matrix derived from the marine organisms is characterized in that within the range of 60 ~ 500nm.
또한, 상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 염증 관련 질환의 치료 및 조직재생을 위해 활용되는 것을 특징으로 한다.In addition, the extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are characterized in that they are utilized for the treatment of inflammation-related diseases and tissue regeneration.
또한, 상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 퇴행성 관절염 질환 치료를 위해 활용되는 것을 특징으로 한다.In addition, the extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are characterized in that they are utilized for the treatment of degenerative arthritis diseases.
또한, 상기 퇴행성 관절염 질환의 치료는 관절에 발생된 염증을 조절하면서 연골조직을 수복하는 것을 특징으로 한다.In addition, the treatment of the degenerative arthritis disease is characterized by repairing cartilage tissue while controlling inflammation generated in the joint.
이상과 같은 구성의 본 발명의 해양생물 유래 세포외 소포의 기능 발현형 약학조성물은 해양생물 특히, 어느 지역에서나 용이하게 확보할 수 있는 해삼으로부터 세포외 소포를 추출하므로 낮은 비용으로 고 기능성 약학조성물을 확보할 수 있는 특징이 있다. The functional expression type pharmaceutical composition of marine life-derived extracellular vesicles of the present invention having the above configuration extracts extracellular vesicles from marine organisms, especially sea cucumbers, which can be easily obtained in any region, so a high-functional pharmaceutical composition at low cost There are features that can be obtained.
또한, 본 발명에 따른 약학조성물은 해양생물 조직으로부터 추출된 세포외 기질로부터 생산되어 기원한 조직의 특성에 따라 다양한 질환 치료 및 조직 재생에 선택적으로 사용될 수 있으므로 효과적인 생물학적 제제 또는 치료제로 활용될 수 있다.In addition, the pharmaceutical composition according to the present invention can be used as an effective biological agent or therapeutic agent because it is produced from the extracellular matrix extracted from marine tissue and can be selectively used for the treatment of various diseases and tissue regeneration depending on the characteristics of the tissue of origin. .
또한, 본 발명에 따른 약학조성물은 해양생물을 분쇄하여 세포외 기질을 추출하고 추출된 세포외 기질에 효소 처리를 통해 높은 수득률로 세포외 소포를 생산할 수 있어 보다 안정적으로 기원한 조직의 특성을 유지하는 세포외 소포를 생산할 수 있다.In addition, the pharmaceutical composition according to the present invention can produce extracellular vesicles in high yield by extracting the extracellular matrix by pulverizing marine organisms and treating the extracted extracellular matrix with enzymes, so more stably maintaining the properties of the originating tissue capable of producing extracellular vesicles.
또한, 본 발명에 따른 약학조성물은 기원한 조직의 특성이 발현되는 세포외 소포와 세포외 기질을 혼합하여 퇴행성 관절염과 같이 염증 치료 및 조직 재생 등 복합적인 치료가 필요한 질환에 적합하도록 응용될 수 있다.In addition, the pharmaceutical composition according to the present invention can be applied to diseases that require complex treatment, such as inflammation treatment and tissue regeneration, such as degenerative arthritis, by mixing extracellular vesicles and extracellular matrix in which the characteristics of the tissue of origin are expressed. .
도 1은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포(SEVs)의 기능 발현형 약학조성물의 제조과정을 도식화한 도면이고,
도 2는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 TEM(도 2A)과 SEM(Scale bar: 0.5um, 도 2B) 이미지이고,
도 3은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 동적 광 산란(Dynamic Light Scattering: DLS) 입자 크기 분석기로 세포외 소포 크기의 유효성을 분석한 그래프와 데이터인데 SEVs의 크기분포는 0.2um필터를 이용해 여과된 탈 이온수에 희석시키고 SEVs 현탁액의 브라운 운동을 이고, DLS(ELS-8000; Ozuka-Electronics, Osaka, Japan)를 사용하여 측정하였다.
도 4는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 형광현미경을 이용한 엔도시토시스(endocytosis)에 대한 이미지이고,
도 5는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 protein cargo의 농도를 막 파괴 전후(SEVs vs. SEVs-lysis)를 대비한 그래프인데 SEVs protein cargo는 용해 완충액(RIPA; Rockland, PA, USA)으로 막을 파괴하고 증류수를 이용해 희석하여 측정하였고, 희석 된 정상 SEVs 및 파괴된 SEVs-lysis는 단백질 검출 시약인 Bradford(Bio-Rad, CA, USA)를 사용하여 파괴 전 후의 단백질 농도를 측정하였다.
도 6은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 농도에 따른 세포독성에 대한 그래프이고,
도 7은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 DAY 0, 1, 3, 5, 7의 농도에 따른 세포증식률에 대한 그래프이고,
도 8은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 7일간 인비트로(in vitro) OA 모델의 염증 마커에 대한 발현도를 나타내는 그래프이다.1 is a diagram schematically illustrating the manufacturing process of a functional expression type pharmaceutical composition of marine life-derived extracellular vesicles (SEVs) according to an embodiment of the present invention;
2 is a TEM (FIG. 2A) and SEM (Scale bar: 0.5um, FIG. 2B) image of a marine organism-derived extracellular vesicle according to an embodiment of the present invention;
3 is a graph and data analyzing the effectiveness of extracellular vesicle size with a dynamic light scattering (DLS) particle size analyzer of marine life-derived extracellular vesicles according to an embodiment of the present invention. The size distribution of SEVs is After dilution in deionized water filtered through a 0.2 μm filter, the Brownian motion of the SEVs suspension was measured using DLS (ELS-8000; Ozuka-Electronics, Osaka, Japan).
4 is an image of endocytosis using a fluorescence microscope of a marine organism-derived extracellular vesicle according to an embodiment of the present invention;
5 is a graph comparing the concentration of protein cargo of marine life-derived extracellular vesicles before and after membrane destruction (SEVs vs. SEVs-lysis) according to an embodiment of the present invention. SEVs protein cargo is a lysis buffer (RIPA; Rockland, PA, USA) was used to destroy the membrane and diluted with distilled water, and the diluted normal SEVs and disrupted SEVs-lysis were measured using Bradford (Bio-Rad, CA, USA), a protein detection reagent, to determine the protein concentration before and after destruction. measured.
6 is a graph of cytotoxicity according to the concentration of marine organisms-derived extracellular vesicles according to an embodiment of the present invention;
7 is a graph of the cell proliferation rate according to the concentrations of
8 is a graph showing the expression level of inflammatory markers in a 7-day in vitro OA model of marine life-derived extracellular vesicles according to an embodiment of the present invention.
이하에서 도면을 참조하여 본 발명에 따른 해양생물 유래 세포외 소포의 기능 발현형 약학조성물에 대해 상세히 설명한다.Hereinafter, with reference to the drawings, the function-expressing pharmaceutical composition of the marine organism-derived extracellular vesicle according to the present invention will be described in detail.
본 발명은 해양생물을 분쇄하여 얻은 조직에서 유래한 세포외 기질(ECM) 생체 재료에서 세포외 소포(extracellular vesicles: EVs)가 풍부하게 유지되어 있는데 착안하여 해양생물 유래 조직에서 ECM을 제조한 후 ECM에서 세포외 소포를 효율적으로 분리 정제하고 분리 정제된 세포외 소포가 기원 조직의 특성이 그대로 발현됨을 실험을 통해 확인하였다. 또한, 항상성이 유지된 상태의 건강한 조직에서 분리한 세포외 소포는 균일한 구성 성분 및 일정한 치료 효과를 보임을 확인하였다.The present invention focuses on the fact that extracellular vesicles (EVs) are abundantly maintained in extracellular matrix (ECM) biomaterials derived from tissues obtained by pulverizing marine organisms. It was confirmed through an experiment that the extracellular vesicles were efficiently separated and purified and that the isolated and purified extracellular vesicles expressed the characteristics of the tissue of origin as they are. In addition, it was confirmed that the extracellular vesicles isolated from healthy tissues in a homeostasis state showed uniform components and a constant therapeutic effect.
본 발명은 해양생물에서 유래된 세포외 기질(Extracellular matrix: ECM)에서 추출한 세포외 소포가 원래 조직에서 가진 기능이 발현되도록 하는 약학조성물을 제공한다.The present invention provides a pharmaceutical composition that enables the function of the extracellular vesicles extracted from the extracellular matrix (ECM) derived from marine organisms to be expressed in the original tissue.
본 발명의 세포외 소포는 해양생물을 분쇄한 후 탈세포화하여 세포외 기질을 수득하고, 수득된 세포외 기질을 효소처리하여 세포외 소포를 얻는다.The extracellular vesicles of the present invention are pulverized and decellularized to obtain an extracellular matrix, and the obtained extracellular matrix is treated with an enzyme to obtain an extracellular vesicle.
보다 상세하게는 수득된 세포외 기질을 PBS로 희석한 collagenase(Worthington, OH, USA) 용액에 담가 실온에서 48시간 동안 교반 시켜 세포외 기질을 모두 파괴시켜 세포외 기질에 붙어있는 세포외 소포를 때어냈다.More specifically, the obtained extracellular matrix is immersed in a collagenase (Worthington, OH, USA) solution diluted with PBS and stirred at room temperature for 48 hours to destroy all the extracellular matrix and break the extracellular vesicles attached to the extracellular matrix. paid
본 발명에 따른 해양생물 유래 세포외 기질로부터 추출된 세포외 소포는 염증을 억제하는 효과를 가질 수 있다. 염증 억제 효과에 대해서는 본 발명의 일실험례를 도 8에 나타내었다.The extracellular vesicles extracted from the marine organism-derived extracellular matrix according to the present invention may have an effect of inhibiting inflammation. As for the anti-inflammatory effect, an experimental example of the present invention is shown in FIG. 8 .
본 발명에 따른 해양생물 유래 세포외 기질로부터 추출된 세포외 소포는 염증과 관련된 질환을 치료하는데 효과가 있고, 예를 들어 퇴행성 관절염과 같이 염증 치료뿐만 아니라 조직재생도 필수적으로 수반되어야 하는 복합적인 치료 과정이 필요한 질환에 특히 효과적이다. 이는 본 발명의 세포외 소포가 조직재생을 위한 세포외 기질과의 혼합이 용이하고 질환의 종류에 따라 염증 치료 외에 다른 치료과정이 필요한 경우 그에 맞는 세포외 기질 등을 혼합하여 단일 약학조성물로 복합치료 효과를 볼 수 있는 특징이 있다. 다만 본 발명의 세포외 소포가 앞서 예로든 퇴행성 관절염 치료에 제한되는 것은 아니다.The extracellular vesicle extracted from the marine organism-derived extracellular matrix according to the present invention is effective in treating inflammation-related diseases, for example, a complex treatment that requires tissue regeneration as well as inflammation treatment, such as degenerative arthritis. It is especially effective for diseases that require a course. This is because the extracellular vesicles of the present invention are easy to mix with the extracellular matrix for tissue regeneration, and if a treatment other than inflammation treatment is required depending on the type of disease, complex treatment with a single pharmaceutical composition by mixing the appropriate extracellular matrix, etc. There are features that can be effective. However, the extracellular vesicles of the present invention are not limited to the treatment of osteoarthritis exemplified above.
<실시예 1> 조직으로부터 세포외 소포 추출<Example 1> Extraction of extracellular vesicles from tissue
도 1은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 기능 발현형 약학조성물의 제조과정을 도식화한 도면이다.1 is a diagram schematically illustrating a manufacturing process of a functional expression type pharmaceutical composition of a marine organism-derived extracellular vesicle according to an embodiment of the present invention.
본 발명의 해양생물 유래된 세포외 기질에서 추출한 세포외 소포는 해삼으로부터 추출하였으며, 크게 2단계로 나눠진다. 먼저 세포외 기질(extracellular matrix; ECM)을 수득한 이후 효소처리를 통해 세포외 소포(extracellular vesicles; EVs)를 추출하는 방법을 사용하였다.The extracellular vesicles extracted from the marine organism-derived extracellular matrix of the present invention were extracted from sea cucumbers, and are largely divided into two steps. First, an extracellular matrix (ECM) was obtained, and then, a method of extracting extracellular vesicles (EVs) through enzyme treatment was used.
우선, 생 해삼의 내장을 제거한 뒤 냉동 보관한다. 이후 200g 냉동 해삼을 잘 용해 될 수 있도록 1~2cm 자른 뒤 0.1M 농도의 저장용액을 이용해 냉장에서 3~4일간 교반 시켜 완전히 용해시켜준다. 3~4일 이후 체를 이용해 완전하게 용해되지 않은 찌꺼기를 걸러준 다음 7,500~8,000xg중력 가속도로 30분간 원심 분리 시킨다. 원심 분리된 ECM은 동결건조과정을 거쳐 스펀지 형태로 만들고 24시간 동한 0.3M HCl 과 pepsin을 이용해 결합을 분해시킨 뒤 NaOH를 이용해 중화 후 24시간 동안 투석하였으며, 완전하게 투석된 해삼 세포외 기질을 동결건조 과정을 통해 추가적으로 스펀지 형태로 제작한다.First, remove the intestines of fresh sea cucumber and store it in the freezer. After that, cut 1~2cm of 200g frozen sea cucumber so that it can be dissolved well, and then use a 0.1M concentration solution and stir in the refrigerator for 3~4 days to completely dissolve it. After 3-4 days, use a sieve to filter out undissolved residues, and then centrifuge for 30 minutes at an acceleration of 7,500-8,000xg gravity. The centrifuged ECM was lyophilized to form a sponge, and the bonds were broken using 0.3M HCl and pepsin for 24 hours, neutralized with NaOH, and dialyzed for 24 hours. Completely dialyzed sea cucumber extracellular matrix was frozen. Through the drying process, it is additionally manufactured in the form of a sponge.
이렇게 수득된 세포외 기질은 세포외 기질을 collagenase 용액에 넣어 실온에서 48시간동안 교반시켜 EVs를 세포외 기질에서 분리 시켰으며, collagenase에 의해 완전하게 분해된 용액을 10,000xg 중력가속도로 1시간 원심 분리 시켜 세포외 기질 찌꺼기를 제거 하였다. 추가적으로 순수한 EVs를 얻기 위해 0.45, 0.2μm 필터를 이용해 잔존해있는 작은 입자또한 제거시켜 주었으며 100kDa 필터와, PEG기반의 상용화 Exoquick(System Biosciences, CA, USA)을 이용해 해삼유래 세포외 소포체를 농축시켰다.EVs were separated from the extracellular matrix by putting the extracellular matrix in a collagenase solution and stirring at room temperature for 48 hours. to remove extracellular matrix debris. Additionally, to obtain pure EVs, small particles remaining were also removed using 0.45 and 0.2 μm filters, and sea cucumber-derived extracellular vesicles were concentrated using a 100 kDa filter and a PEG-based commercial Exoquick (System Biosciences, CA, USA).
보다 상세하게는 탈세포화된 세포외 기질은 콜라게나제 I(collagenase I), 콜라게나제 II(collagenase II), 콜라게나제 B(collagenase B), 프로테이나제 K(Proteinase-K) 및 펩신(Pepsin)으로 이루어진 군에서 선택되는 하나 이상의 효소를 처리하여 용해될 수 있다.More specifically, the decellularized extracellular matrix is collagenase I (collagenase I), collagenase II (collagenase II), collagenase B (collagenase B), proteinase K (Proteinase-K) and pepsin (Pepsin) can be dissolved by treating one or more enzymes selected from the group consisting of.
<실시예 2>세포외 소포의 특성 분석<Example 2> Characterization of extracellular vesicles
도 2는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 TEM(도 2A)과 SEM(Scale bar: 0.5um, 도 2B) 이미지인데, 전자현미경을 이용하여 이미지를 얻었으며 투과 전자 현미경(TEM)(H-7500; HITACHI, Tokyo, Japan) 및 주사 전자 현미경(SEM)(JEM-2100F; JEOL, Japan)으로 수행되었다. SEVs를 2.5 % 농도의 글루 타르 알데히드(JUNSEI, Tokyo, Tokyo, Japan)에 24 시간 동안 고정하고 소량의 SEVs 혼합물을 탄소 코팅 그리드(TED PELLA, CA, USA)에 떨어뜨린 후 실온에서 건조시키고 TEM으로 촬영 하였으며 cover glass 위에서 건조시킨 뒤 SEM으로 촬영하였다.2 is a TEM (FIG. 2A) and SEM (Scale bar: 0.5um, FIG. 2B) images of marine life-derived extracellular vesicles according to an embodiment of the present invention. The images were obtained using an electron microscope and transmission electron microscope (TEM) (H-7500; HITACHI, Tokyo, Japan) and scanning electron microscopy (SEM) (JEM-2100F; JEOL, Japan). SEVs were fixed in 2.5% concentration of glutaraldehyde (JUNSEI, Tokyo, Tokyo, Japan) for 24 h, a small amount of the SEVs mixture was dropped on a carbon-coated grid (TED PELLA, CA, USA), dried at room temperature, and TEM It was photographed, dried on a cover glass, and then photographed by SEM.
도 3은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 동적 광 산란(Dynamic Light Scattering: DLS) 입자 크기 분석기로 세포외 소포 크기의 유효성을 분석한 그래프와 데이터인데 SEVs의 크기분포는 0.2um필터를 이용해 여과된 탈 이온수에 희석시키고 SEVs 현탁액의 브라운 운동을 이고, DLS(ELS-8000; Ozuka-Electronics, Osaka, Japan)를 사용하여 측정하였다.3 is a graph and data analyzing the effectiveness of extracellular vesicle size with a dynamic light scattering (DLS) particle size analyzer of marine life-derived extracellular vesicles according to an embodiment of the present invention. The size distribution of SEVs is After dilution in deionized water filtered through a 0.2 μm filter, the Brownian motion of the SEVs suspension was measured using DLS (ELS-8000; Ozuka-Electronics, Osaka, Japan).
도 4는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 형광현미경을 이용한 엔도시토시스(endocytosis)에 대한 이미지인데, SEVs의 막을 PKH-26(Sigma-Aldrich, MO, USA) 세포막 염색 시약을 이용해 4℃에서 24 시간 동안 표지 하였으며 표지 된 SEVs를 0.2 μm 여과 된 PBS로 세척하고 100k Da 필터를 사용하여 다시 농축시켜 SEVs를 수득 하였다. 이후 세포내 흡수를 관찰하기 위해 10 % exosome deplete 소 태아 혈정(FBS; Gibco, MA, USA) 및 1 % 항생제 (Gibco, MA, USA)를 함유하는 DMEM(GE Healthcare, MA, USA)에서 희석시킨 SEVs를 활막 세포에 6 시간 처리한 뒤, DAPI 형광 시약(Thermo, MA, USA) 을 처리하고 형광 현미경(Axio-Observer 5; Carl Zeiss, Overkochen, Germany)을 사용하여 세포 내로의 흡수됨을 관찰 하였다.4 is an image of endocytosis using a fluorescence microscope of marine life-derived extracellular vesicles according to an embodiment of the present invention. SEVs membranes are stained with PKH-26 (Sigma-Aldrich, MO, USA) cell membrane. The reagent was labeled at 4 °C for 24 hours, and the labeled SEVs were washed with 0.2 μm filtered PBS and concentrated again using a 100 k Da filter to obtain SEVs. Then, to observe intracellular uptake, it was diluted in DMEM (GE Healthcare, MA, USA) containing 10% exosome deplete fetal bovine serum (FBS; Gibco, MA, USA) and 1% antibiotic (Gibco, MA, USA). After SEVs were treated with synovial cells for 6 hours, DAPI fluorescence reagent (Thermo, MA, USA) was treated, and absorption into the cells was observed using a fluorescence microscope (Axio-
도 2의 TEM, SEM 이미지를 참조하면 본 발명의 세포외 소포가 해삼 조직의 물리적 특성인 원형의 형태를 갖고 있음을 알 수 있고, 도 3의 본 발명의 세포외 소포의 크기 분포가 해삼 조직의 크기 분포와 패턴이 동일하고, 도 4의 이미지를 참조하면 본 발명의 세포외 소포가 해삼의 세포외 소포와 엔도시토시스가 동일함을 알 수 있고, 본 발명의 일실시예로 제조된 세포외 소포가 해삼에서 유래되어 그대로 유지되고 있음을 알 수 있는데 도 4의 이미지를 통해 추출과정에서 해삼의 석회성분이 모두 제거 되었고 추가적으로 세포 내로 흡수될 때 세포외 소포의 모양이 변형되지 않음을 알 수 있다.Referring to the TEM and SEM images of FIG. 2 , it can be seen that the extracellular vesicles of the present invention have a circular shape, which is a physical characteristic of the sea cucumber tissue, and the size distribution of the extracellular vesicles of the present invention in FIG. 3 is that of the sea cucumber tissue. The size distribution and pattern are the same, and referring to the image of FIG. 4 , it can be seen that the extracellular vesicles of the present invention have the same endocytosis as the extracellular vesicles of sea cucumber, and the extracellular vesicles prepared according to an embodiment of the present invention It can be seen that the vesicle is derived from sea cucumber and is maintained as it is. Through the image of FIG. 4 , all the lime components of the sea cucumber were removed during the extraction process, and it can be seen that the shape of the extracellular vesicle is not deformed when it is additionally absorbed into the cell. .
<실시예 3>세포외 소포의 protein cargo 함유량 분석<Example 3> Analysis of protein cargo content of extracellular vesicles
도 5는 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 protein cargo의 농도를 SEVs-lysis와 대비한 그래프이다.5 is a graph comparing the concentration of protein cargo of marine organisms-derived extracellular vesicles with SEVs-lysis according to an embodiment of the present invention.
일반적으로 세포외 소포는 기원과 분비 기작, 크기 등을 기준으로 엑소좀(exosomes), 마이크로베시클(microvesicles), 엑토좀(ectosomes) 등 다양한 명칭으로 불리는데 세포외 소포는 단백질, 지질, 핵산, 대사물질(metabolites) 등 생물학적 활성을 보이는 물질을 포함하고 있다. 도 5를 통해 본 발명의 세포외 소포의 막 파괴 전후의 protein cargo의 농도를 통해 본 발명의 세포외 소포 내부에 단백질이 다량 존재하고 있음을 알 수 있다.In general, extracellular vesicles are called by various names such as exosomes, microvesicles, and ectosomes based on their origin, secretion mechanism, and size. It contains substances that exhibit biological activity, such as metabolites. 5, it can be seen that a large amount of protein is present inside the extracellular vesicle of the present invention through the concentration of protein cargo before and after membrane destruction of the extracellular vesicle of the present invention.
<실시예 4>세포외 소포의 세포 독성 및 세포 증식률<Example 4> Cytotoxicity and cell proliferation rate of extracellular vesicles
도 6은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 농도에 따른 세포독성에 대한 그래프인데, 다양한 농도의 SEVs (0, 1, 5, 10, 20 μg / ml)를 활막 세포 2x104 농도 에서 24시간동안 37 ℃ 및 5 % CO2 인큐베이터 에서 배양 한 후 세포 독성 실험을 수행 하였으며 SEVs처리에 따른 미토콘드리아 활성에 대한 WST-assay 시약(EZ-cytox, DoGenBio, South Korea)을 이용하였으며 450nm의 파장의 흡광도를 plate reader기(Molecular Device, CA ,USA)를 이용하여 측정하였다.6 is a graph of cytotoxicity according to the concentration of marine life-derived extracellular vesicles according to an embodiment of the present invention, wherein SEVs (0, 1, 5, 10, 20 μg / ml) of various concentrations were treated with 2x104 synovial cells. After culturing in an incubator at 37 °C and 5% CO2 for 24 hours at a concentration of , a cytotoxicity experiment was performed, and a WST-assay reagent (EZ-cytox, DoGenBio, South Korea) for mitochondrial activity according to SEVs treatment was used and a wavelength of 450 nm The absorbance was measured using a plate reader (Molecular Device, CA, USA).
도 7은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 DAY 0, 1, 3, 5, 7의 농도에 따른 세포증식률에 대한 그래프인데 다양한 농도의 SEV (0, 5, 10, 20 μg / ml)를 활막 세포 5x103 농도에서 1, 3, 5, 7 일 동안 도 6과 동일한 조건인 37 ℃ 및 5 % CO2 인큐베이터 에서 배양 한 후 세포 증식률에 미치는 영향에 관한 실험을 진행 하였다. 세포독성 검사와 동일하게 SEVs 처리에 따른 미토콘드리아 활성의 WST-assay 시약(EZ-cytox, DoGenBio, South Korea)을 이용하였으며 450nm의 파장의 흡광도를 plate reader기 (Molecular Device, CA ,USA) 를 이용해 측정하였다.7 is a graph showing the cell proliferation rate according to the concentration of
SEVs를 처리한 모든 그룹에서 3일째에 감소하는 것처럼 보였으나 5, 7 일째 결과에서 볼 수 있듯이 세포독성에 따른 증식률 변화는 관찰되지 않았다.All groups treated with SEVs seemed to decrease on the 3rd day, but as can be seen from the results on the 5th and 7th days, no change in the proliferation rate according to cytotoxicity was observed.
<실시예 5>세포외 소포의 염증 억제 효과<Example 5> Inhibitory effect of extracellular vesicles on inflammation
도 8은 본 발명의 일실시예에 따른 해양생물 유래 세포외 소포의 7일간 인비트로(in vitro) OA 모델의 염증 마커에 대한 발현도를 나타내는 그래프로 SEVs 처리에 따른 in vitro OA모델 유전자 발현변화에 관한 결과이다. 8 is a graph showing the expression level of inflammatory markers in a marine organism-derived extracellular vesicle in an in vitro OA model for 7 days according to an embodiment of the present invention. is a result about
IL-1β는 연골의 주요 성분인 collagen type 2 와 GAG 합성을 억제하고 통증을 유발시키는 요소이고, IL-6은 ECM분해에 관여하는 MMP(Matrix metalloproteinase) 발현을 증가시키고 collagen type 2 발현을 감소시키는 것과 관련이 있고, 마지막으로 COX-2는 염증, 발열, 통증을 유발하는 프로스타글란딘-2 합성에 관여하는 효소이므로 골 관절염과 같은 만성 질환에서 발생하는 통증에 중요한 역할을 하기 때문에 대표적으로 4가지 유전 물질 발현 양을 비교하였습니다. IL-1β inhibits the synthesis of
IL-1β 그래프에서 보는 것처럼 SEVs를 처리한 그룹에서 관련 유전 물질이 통계적으로 유의미한 정도 까지 감소하였으며 normal 그룹과 유사한 정도의 유전자 발현이 관찰이 되었으며 IL-6 결과 또한 inflammation 그룹보다 감소함을 확인 하였다. 그리고 SEVs 처리 군에서 MMP-1 그리고 COX-2 유전자의 양은 정상 세포 유전자 수준으로 확연하게 감소한 것을 확인하였다.As shown in the IL-1β graph, in the group treated with SEVs, the related genetic material decreased to a statistically significant level, and gene expression similar to that of the normal group was observed, and it was confirmed that the IL-6 result was also decreased compared to the inflammation group. And it was confirmed that the amount of MMP-1 and COX-2 genes in the SEVs-treated group was significantly reduced to the normal cell gene level.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It is clear. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (10)
A functional expression type pharmaceutical composition of extracellular vesicles derived from marine organisms, comprising extracellular vesicles extracted from extracellular matrix derived from marine organisms as an active ingredient.
상기 세포외 기질에서 추출한 세포외 소포는 해상 생물 중 해삼으로부터 추출된 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
The extracellular vesicles extracted from the extracellular matrix are functional expression-type pharmaceutical composition of marine organisms-derived extracellular vesicles, characterized in that they are extracted from sea cucumbers among marine organisms.
해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 엑소좀인 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
A function-expressing pharmaceutical composition for extracellular vesicles derived from marine organisms, characterized in that the extracellular vesicles extracted from the extracellular matrix derived from marine organisms are exosomes.
상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 해양동물을 분쇄하여 세포외기질을 추출하고,
상기 추출된 세포외기질에 효소처리 단계를 거쳐 세포외 소포를 추출하는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
The extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are pulverized to extract the extracellular matrix,
A functional expression type pharmaceutical composition of marine organisms-derived extracellular vesicles, characterized in that the extracted extracellular matrix is subjected to an enzyme treatment step to extract the extracellular vesicles.
상기 세포외기질이 추출은 분쇄된 해양동물 조직을 교반하는 단계를 통해 해양동물 조직을 용해시키는 단계, 용해되지 않은 찌꺼기를 걸러주는 단계와 원심분리하는 단계를 통해 추출하는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 4,
The extraction of the extracellular matrix is derived from marine organisms, characterized in that the extraction is carried out through the steps of dissolving the marine animal tissue through agitation of the pulverized marine animal tissue, filtering the undissolved debris, and centrifuging A functional expression type pharmaceutical composition of extracellular vesicles.
상기 추출된 세포외기질에 효소처리하는 단계는 추출된 세포외기질을 동결건조한 후 HCL과 펩신을 이용하여 세포외기질의 결합을 분해하는 단계, NaOH를 이용하여 중화시킨 후 투석하는 단계, 중화 투석된 세포외기질을 collagenase를 첨가한 후 교반하는 단계 및 원심 분리를 통해 찌꺼기를 제거하는 단계를 통해 세포외 소포를 추출하는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 4,
The enzymatic treatment of the extracted extracellular matrix includes lyophilizing the extracted extracellular matrix and then using HCL and pepsin to break the binding of the extracellular matrix, neutralizing it using NaOH and then dialysis, neutralizing dialysis A functional expression type pharmaceutical composition for extracellular vesicles derived from marine organisms, characterized in that the extracellular vesicles are extracted through the steps of agitating the extracellular matrix after adding collagenase and removing the debris through centrifugation.
상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포의 크기는 60~600nm 범위 내에 있는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
The functional expression type pharmaceutical composition of extracellular vesicles derived from marine organisms, characterized in that the size of the extracellular vesicles extracted from the extracellular matrix derived from the marine organisms is within the range of 60 to 600 nm.
상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 염증 관련 질환의 치료 및 조직재생을 위해 활용되는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
The extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are used for the treatment of inflammation-related diseases and tissue regeneration, a functional expression type pharmaceutical composition of the extracellular vesicles derived from marine organisms.
상기 해양생물에서 유래된 세포외 기질에서 추출한 세포외 소포는 퇴행성 관절염 질환 치료를 위해 활용되는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 1,
The extracellular vesicles extracted from the extracellular matrix derived from the marine organisms are used for the treatment of degenerative arthritis diseases.
상기 퇴행성 관절염 질환의 치료는 관절에 발생된 염증을 조절하면서 연골조직을 수복하는 것을 특징으로 하는 해양생물 유래 세포외 소포의 기능 발현형 약학조성물.
In claim 9,
The treatment of the degenerative arthritis disease is a marine organism-derived extracellular vesicle function expression type pharmaceutical composition, characterized in that it restores cartilage tissue while controlling inflammation generated in the joint.
Priority Applications (1)
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