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KR20210005397A - Liposomal nanoparticle and composition for treating myocardial infarction containing the same - Google Patents

️Thu Jan 14 2021

Liposomal nanoparticle and composition for treating myocardial infarction containing the same Download PDF

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Publication number

KR20210005397A

KR20210005397A KR1020190080623A KR20190080623A KR20210005397A KR 20210005397 A KR20210005397 A KR 20210005397A KR 1020190080623 A KR1020190080623 A KR 1020190080623A KR 20190080623 A KR20190080623 A KR 20190080623A KR 20210005397 A KR20210005397 A KR 20210005397A Authority

South Korea

Prior art keywords

liposome

antigen

nanoparticles

myocardial infarction

dendritic cells

Prior art date

2019-07-04

Application number

KR1020190080623A

Other languages

Korean (ko)

Other versions

KR102283256B1 (en

Inventor

장기육

강권윤

박은혜

김병수

권성필

Original Assignee

가톨릭대학교 산학협력단

서울대학교산학협력단

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2019-07-04

Filing date

2019-07-04

Publication date

2021-01-14

2019-07-04 Application filed by 가톨릭대학교 산학협력단, 서울대학교산학협력단 filed Critical 가톨릭대학교 산학협력단

2019-07-04 Priority to KR1020190080623A priority Critical patent/KR102283256B1/en

2021-01-14 Publication of KR20210005397A publication Critical patent/KR20210005397A/en

2021-07-30 Application granted granted Critical

2021-07-30 Publication of KR102283256B1 publication Critical patent/KR102283256B1/en

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Abstract

본 발명은 내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자 및 이의 심근경색 치료 용도에 관한 것이다. The present invention relates to liposome nanoparticles carrying an antigen in an inner compartment and carrying an immunotolerant in a double lipid membrane, and a use thereof for treating myocardial infarction.

Description

리포좀 나노입자 및 이를 포함하는 심근경색 치료용 조성물{LIPOSOMAL NANOPARTICLE AND COMPOSITION FOR TREATING MYOCARDIAL INFARCTION CONTAINING THE SAME}Liposomal nanoparticles and compositions for treatment of myocardial infarction containing the same TECHNICAL FIELD {LIPOSOMAL NANOPARTICLE AND COMPOSITION FOR TREATING MYOCARDIAL INFARCTION CONTAINING THE SAME}

본 발명은 내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자 및 이의 심근경색 치료 용도에 관한 것이다.The present invention relates to liposome nanoparticles carrying an antigen in an inner compartment and carrying an immunotolerant in a double lipid membrane, and a use thereof for treating myocardial infarction.

심근경색은 심장혈관이 혈전, 연축 등의 원인에 의해 갑자기 막혀서 심장 근육이 손상되는 질환으로서, 4, 50대 성인 돌연사의 원인 1위를 차지한다. 심근경색은 심장의 관상동맥 혈관내강이 콜레스테롤과 임파구의 괴사덩어리인 동맥경화반의 축적과 성장으로, 병변이 성장하면서 혈관내강이 좁아져 생기는 관상동맥질환(협심증 등)으로부터 유래되나, 병변의 섬유성막(fibrous cap)이 매우 불안정하여 쉽게 병변의 파열(rupture)이 일어나고 혈전의 형성이 일어나 혈관의 내강이 완전히 막히는 현상(thrombosis)으로 인하여 생긴다. 일단 심근경색이 발생하면 그 혈관의 하위지점에 있는 근육들은 산소와 영양이 공급되지 않아 심장근육의 괴사가 일어난다. 따라서, 심근경색은 죽상경화로 시작되어 장기적으로 진행 발병하나, 병변이 안정적으로 형성되어 섬유성막이 쉽게 파열되지 않는 협심증(angina pectoris)과 달리, 병변의 형성과 파열이 매우 쉽게 진행되는 취약성 동맥경화반(vulnerable plaque)의 형성이라는 점에서 협심증과 차이가 있다.Myocardial infarction is a disease in which cardiac blood vessels are suddenly blocked by causes such as blood clots and spasms and damages the heart muscle, and occupies the number 1 cause of sudden death in adults in their 4's and 50s. Myocardial infarction is derived from coronary artery disease (angina, etc.), which is caused by the accumulation and growth of arteriosclerotic plaques, which are necrotic masses of cholesterol and lymphocytes, in the coronary artery of the heart, and narrowing the vascular lumen as the lesion grows. The fibrous cap is very unstable, so the lesion is easily rupture and the formation of blood clots occurs due to thrombosis of the blood vessel lumen. Once a myocardial infarction occurs, the muscles in the lower points of the blood vessel are not supplied with oxygen and nutrition, resulting in necrosis of the heart muscle. Therefore, myocardial infarction begins with atherosclerosis and progresses for a long time, but unlike angina pectoris in which the fibrous membrane is not easily ruptured due to the stable formation of the lesion, the fragile arteriosclerosis in which the formation and rupture of the lesion proceeds very easily. It differs from angina in that it forms a vulnerable plaque.

심근 경색 후의 사망은 경색 발생 수 시간 이내에 가장 많이 일어나는데, 1시간 이내에 약 50%의 환자가 사망하고, 24시간 이내에 약 80%의 환자가 사망하는 것으로 알려져 있다. 그러므로 심근경색의 치료가 빨리 시작될수록 심근 경색에 의한 환자의 사망률을 줄일 수 있다.Death after myocardial infarction occurs most frequently within a few hours of infarction, and it is known that about 50% of patients die within 1 hour and about 80% of patients die within 24 hours. Therefore, the sooner the treatment of myocardial infarction is started, the less the patient's mortality due to myocardial infarction can be reduced.

또한, 심근경색증 발생 후 재발을 예방함으로써 심근경색증 후 장기 사망률과 이환율을 줄일 수 있다. 심근경색의 재발을 예방하는 약물로는 아스피린, 베타차단제, ACE 저해제, 항혈전제 등이 알려져 있다. 그러나 아스피린의 경우 알러지, 내성 등의 문제점이 있으며, 항혈전제의 경우에는 출현 위험성이 증가하는 문제점이 있다.In addition, it is possible to reduce long-term mortality and morbidity after myocardial infarction by preventing recurrence after myocardial infarction. Drugs that prevent recurrence of myocardial infarction include aspirin, beta-blockers, ACE inhibitors, and antithrombotic agents. However, in the case of aspirin, there are problems such as allergy and resistance, and in the case of antithrombotic drugs, there is a problem that the risk of appearance increases.

경색 발병 시에 해당 부근에서 일시적인 염증성 인자 및 활성산소의 발생량이 증가하여 주변 심근세포에 피해를 준다. 뿐만 아니라 T 세포나 대식세포와 같은 면역세포를 자극하여 이로 인해 과도한 염증 반응이 지속적으로 유도되어 심근경색의 증상을 악화시킨다. 과도한 염증 반응을 억제하기 위한 염증 조절반응은 심근경색의 치료에 중요요소로서 주로 조절 T 세포에 의해 작동된다. 조절 T 세포는 경색 부위에 분포한 자극된 T 세포나 대식세포를 억제함과 동시에 항염증성 인자를 분비하여 심장에서의 염증 반응을 완화시킨다.At the onset of infarction, the amount of transient inflammatory factors and free radicals are increased in the vicinity, causing damage to surrounding cardiomyocytes. In addition, by stimulating immune cells such as T cells or macrophages, excessive inflammatory reactions are continuously induced, which worsens symptoms of myocardial infarction. The inflammatory modulatory response to suppress the excessive inflammatory response is an important factor in the treatment of myocardial infarction and is mainly driven by regulatory T cells. Regulatory T cells suppress the stimulated T cells or macrophages distributed in the infarct area and at the same time secrete anti-inflammatory factors to relieve the inflammatory response in the heart.

심근경색을 치료하기 위한 방법으로써 대표적으로 줄기세포, 조절 T 세포, 조절 수지상 세포를 이용한 방법이 개발되고 있다. 줄기세포를 이용한 방법은 많은 연구가 되었으나, 임상치료효과가 극히 미미하며, 면역학적 측면에서 질환의 진행을 억제하지 못한다. 대안으로 조절 T 세포와 조절 수지상 세포가 연구되고 있으나, 수율 확보의 어려움과 불분명한 주입경로, 높은 가격과 같은 문제들로 인해 여전히 임상적용에 어려움을 갖고 있다. 또한 심근경색의 특성상 발병 후 최대한 빠른 시간내에 치료가 이루어져야 한다는 면에서 준비시간이 긴 세포치료제는 적절한 치료법이 되지 못한다. 이를 대체하기 위한 기술로써 환자 내원 즉시 치료가능하고 가격이 저렴한 나노입자를 이용하여 체내에서 조절 수지상 세포, 조절 T 세포를 유도하는 기술의 개발이 요구되고 있다.As a method for treating myocardial infarction, a method using stem cells, regulatory T cells, and regulatory dendritic cells has been developed. The method using stem cells has been studied a lot, but the clinical treatment effect is very insignificant, and it cannot suppress the progression of the disease in terms of immunological aspects. As an alternative, regulatory T cells and regulatory dendritic cells are being studied, but they still have difficulty in clinical application due to problems such as difficulty in securing yield, unclear injection route, and high price. In addition, due to the nature of myocardial infarction, cell therapy products with a long preparation time are not adequate treatment in that treatment must be performed within the shortest possible time after onset. As a technology to replace this, there is a need to develop a technology for inducing regulatory dendritic cells and regulatory T cells in the body using nanoparticles that can be treated immediately and inexpensively on patient visits.

한국등록특허 제10-1628453호Korean Patent Registration No. 10-1628453

이에 본 발명자들은 내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자를 체내에 투여하였을 때 면역관용 수지상 세포를 성숙시켜 Treg 세포를 활성화시킴으로써 심근경색 및 자가면역질환을 치료할 수 있음을 발견함으로써 본 발명을 완성하였다.Accordingly, the present inventors can treat myocardial infarction and autoimmune diseases by activating Treg cells by maturing immune-tolerant dendritic cells when liposomal nanoparticles carrying antigens in the inner compartment and immunotolerant in the lipid membrane are administered into the body. The present invention was completed by finding that there is.

상기와 같은 목적을 달성하기 위하여, 본 발명은 내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자를 제공한다.In order to achieve the above object, the present invention provides a liposome nanoparticle in which an antigen is supported in an inner compartment and an immunotolerant is supported in a double lipid membrane.

또한, 본 발명은 본 발명에 따른 리포좀 나노입자를 포함하는 크론병, 루푸스, 제1당뇨병, 류마티스 관절염, 이식편대숙주질환(GvHD), 알러지, 천식, 간염, 궤양성 대장염, 혈우병, 골 관절염, 척수손상질환, 뇌졸중 또는 심근경색 치료용 조성물을 제공한다.In addition, the present invention is Crohn's disease, lupus, first diabetes, rheumatoid arthritis, graft versus host disease (GvHD), allergies, asthma, hepatitis, ulcerative colitis, hemophilia, osteoarthritis, including liposome nanoparticles according to the present invention. It provides a composition for treating spinal cord injury, stroke or myocardial infarction.

본 발명의 리포좀 나노입자는 체내에 투여됐을 때 림프절을 표적화하여 림프절에 전달되어 면역관용 수지상 세포를 성숙시켜 Treg 세포를 활성화시킴으로써 심장의 면역 반응을 조절하여 심근경색 후 심장의 기능을 회복하고 경색의 크기를 감소시켜 심근경색의 치료에 효과적으로 활용될 수 있다. 또한, 본 발명의 리포좀 나노입자는 자가면역 항원 및 면역관용물질을 체내에 전달하여 자가면역질환을 치료할 수 있다.When the liposome nanoparticles of the present invention are administered into the body, the liposomal nanoparticles of the present invention target lymph nodes and are delivered to the lymph nodes to mature immune-tolerant dendritic cells to activate Treg cells, thereby regulating the immune response of the heart, restoring heart function after myocardial infarction By reducing the size, it can be effectively used in the treatment of myocardial infarction. In addition, the liposome nanoparticles of the present invention can treat autoimmune diseases by delivering autoimmune antigens and immune tolerant substances into the body.

도 1은 본 발명의 일 구현예에 따른 리포좀 나노입자의 작용 메카니즘을 도시한 것이다.
도 2는 본 발명의 일 구현예에 따른 리포좀 나노입자의 제조공정을 나타낸 것이다.
도 3은 본 발명의 일 실시예에 따른 리포좀 나노입자의 형태, 지름 및 TEM 이미지를 나타낸 것이다.
도 4a는 본 발명의 일 실시예에 있어서 리포좀 나노입자에 라파마이신 및 항원이 담지된 것을 형광으로 확인한 결과를 나타낸 것이다.
도 4b는 본 발명의 일 실시예에 있어서 수용액 내에서의 라파마이신 및 라파마이신이 담지된 리포좀을 나타낸 것이다.
도 4c는 본 발명의 일 실시예에 있어서 라파마이신, 라파마이신이 담지된 리포좀 및 항원과 라파마이신이 담지된 리포좀의 DSC 열분석 결과를 나타낸 그래프이다.
도 4d는 본 발명의 일 실시예에 있어서 리포좀에 라파마이신이 담지됨을 확인하는 UV 스펙트럼 결과이다.
도 5는 본 발명의 일 실시예에 있어서 리포좀이 수지상 세포로의 uptake 효율을 확인한 결과이다.
도 6a는 본 발명의 일 실시예에 있어서 리포좀의 항원 전달 효율성을 유세포 분석기로 확인한 결과이다.
도 6b는 본 발명의 일 실시예에 있어서 리포좀의 항원 전달 및 수지상 세포로의 항원표출을 공초점 현미경으로 확인한 결과이다.
도 7a는 본 발명의 일 실시예에 있어서 리포좀의 면역관용 수지상 세포 유도 능력을 확인하기 위해 유세포 분석기로 공동자극분자 발현량을 확인한 결과이다.
도 7b는 본 발명의 일 실시예에 있어서 리포좀의 면역관용 수지상 세포 유도 능력을 확인하기 위해 중합효소연쇄반응으로 mRNA 발현량을 확인한 결과이다.
도 7c는 본 발명의 일 실시예에 있어서 리포좀의 면역관용 수지상 세포 유도 능력을 확인하기 위해 입자처리된 수지상 세포와 CD4 T 세포와 공배양하여 T세포 증식 억제능력을 확인한 결과이다.
도 8a는 본 발명의 일 실시예에 있어서 리포좀에 의해 유도된 면역관용 수지상 세포가 조절 T세포를 유도시키는 지의 유무를 확인하기 위해 유세포 분석기 및 중합효소연쇄반응을 통해 관련 인자 발현을 확인한 결과이다.
도 8b는 본 발명의 일 실시예에 있어서 리포좀에 의해 유도된 면역관용 수지상 세포에 의해 생성된 조절 T세포의 세포 성질을 확인하기 위해 중합효소연쇄반응을 실행한 결과를 나타낸 것이다.
도 9은 본 발명의 일 실시예에 있어서 리포좀 나노입자가 림프절을 표적화하는지 확인하기 위한 실험 결과를 나타낸 것이다.
도 10은 본 발명의 일 실시예에 있어서 리포좀 나노입자의 심근경색 치료 효과를 검증하기 위한 실험 디자인을 도시한 것이다.
도 11a은 본 발명의 일 실시예에 있어서 리포좀 나노입자의 투여 후 심초음파 결과를 나타낸 것이다.
도 11b는 본 발명의 일 실시예에 있어서 리포좀 나노입자의 투여 후 심장 조직의 MT stain 결과를 나타낸 것이다.
도 12는 본 발명의 일 실시예에 있어서 리포좀 나노입자의 투여 후 심장조직 내 M1/M2 대식세포 변화를 나타낸 것이다.1 shows a mechanism of action of liposome nanoparticles according to an embodiment of the present invention.
Figure 2 shows the manufacturing process of the liposome nanoparticles according to an embodiment of the present invention.
3 shows the shape, diameter, and TEM images of liposome nanoparticles according to an embodiment of the present invention.
Figure 4a shows the result of confirming by fluorescence that rapamycin and an antigen are supported on liposome nanoparticles in an embodiment of the present invention.
Figure 4b shows rapamycin and rapamycin-supported liposomes in an aqueous solution according to an embodiment of the present invention.
4C is a graph showing DSC thermal analysis results of rapamycin, liposomes carrying rapamycin, and liposomes carrying antigen and rapamycin according to an embodiment of the present invention.
Figure 4d is a UV spectrum result confirming that rapamycin is supported on the liposome in an embodiment of the present invention.
5 is a result of confirming the uptake efficiency of liposomes to dendritic cells in an embodiment of the present invention.
6A is a result of confirming the antigen delivery efficiency of liposomes by flow cytometry in an embodiment of the present invention.
6B is a result of confirming the antigen delivery of liposomes and antigen expression to dendritic cells with a confocal microscope in an embodiment of the present invention.
7A is a result of confirming the expression level of co-stimulatory molecules by flow cytometry to confirm the ability of liposomes to induce immune-tolerant dendritic cells in an embodiment of the present invention.
7B is a result of confirming the mRNA expression level by polymerase chain reaction in order to confirm the ability of liposomes to induce immune-tolerant dendritic cells in an embodiment of the present invention.
7C is a result of confirming the ability to inhibit T cell proliferation by co-culture with particle-treated dendritic cells and CD4 T cells to confirm the ability of liposomes to induce immune-tolerant dendritic cells in an embodiment of the present invention.
Figure 8a is a result of confirming the expression of related factors through flow cytometry and polymerase chain reaction in order to confirm whether or not immune-tolerant dendritic cells induced by liposomes induce regulatory T cells in an embodiment of the present invention.
Figure 8b shows the results of performing a polymerase chain reaction to confirm the cellular properties of regulatory T cells produced by immunotolerant dendritic cells induced by liposomes in an embodiment of the present invention.
9 shows the experimental results for confirming whether the liposome nanoparticles target lymph nodes in an embodiment of the present invention.
10 shows an experimental design for verifying the myocardial infarction treatment effect of liposome nanoparticles in an embodiment of the present invention.
Figure 11a shows the echocardiographic results after administration of liposome nanoparticles in an embodiment of the present invention.
Figure 11b shows the MT staining results of the heart tissue after administration of the liposome nanoparticles in an embodiment of the present invention.
12 shows the change in M1/M2 macrophages in heart tissue after administration of liposome nanoparticles in an embodiment of the present invention.

이하, 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 이에 의해 본 발명이 제한되지는 않으며 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail as an embodiment of the present invention. However, the following embodiments are presented as examples of the present invention, whereby the present invention is not limited, and the present invention can be variously modified and applied within the scope of equality interpreted from the description of the claims to be described later. .

본 발명에 있어서 용어, "수지상 세포 (dendritic cell)" 는 항원을 세포 내부로 흡수하여 MHC 클래스 I 복합체 또는 MHC 클래스 II 복합체와 함께 다양한 항원 샘플을 T 세포에 제시하는 전문적 항원 제시 세포를 말한다. 수지상 세포는 성숙도 또는 표면 표현형의 발현 정도에 따라 미성숙 수지상 세포 (immature dendritic cell), 준성숙 수지상 세포 (semi-mature dendritic cells) 및 성숙 수지상 세포(mature dendritic cell)로 분류할 수 있다. 수지상 세포의 표면 마커의 발현 프로파일링은 당업계에 공지된 유세포 분석을 통하여 용이하게 할 수 있다.In the present invention, the term "dendritic cell" refers to a specialized antigen-presenting cell that absorbs an antigen into a cell and presents various antigen samples together with an MHC class I complex or an MHC class II complex to T cells. Dendritic cells can be classified into immature dendritic cells, semi-mature dendritic cells, and mature dendritic cells according to the degree of maturity or expression of the surface phenotype. Expression profiling of the surface markers of dendritic cells can be facilitated through flow cytometry known in the art.

본 발명에 있어서 용어, "미성숙 수지상 세포" 는 수지상 세포의 초기 성숙 단계에 발견되는 것으로, 단핵구 세포의 표면 표현형인 CD14 를 발현하지 않으며, 공동 자극 분자인 CD40, CD80 및 CD86을 낮은 수준으로 발현하는 수지상 세포를 말한다.In the present invention, the term "immature dendritic cells" is found in the early maturation stage of dendritic cells, does not express CD14, a surface phenotype of monocytes, and expresses co-stimulatory molecules CD40, CD80 and CD86 at low levels. Refers to dendritic cells.

본 발명에 있어서 용어, "성숙 수지상 세포" 는 미성숙 수지상 세포가 성숙화되어 형성된 세포를 의미한다. 성숙 수지상 세포는 MHC 클래스 II, CD40, CD80 및 CD86의 증가된 발현을 나타내며, 친-염증성 사이토카인(proinflammatory cytokine)을 방출하며, 원시 T 세포 (naive T cell)를 활성화시켜 면역반응을 유도할 수 있는 능력을 가진다.In the present invention, the term "mature dendritic cells" means cells formed by maturation of immature dendritic cells. Mature dendritic cells show increased expression of MHC class II, CD40, CD80, and CD86, release proinflammatory cytokines, and can induce immune responses by activating naive T cells. Have the ability to

본 발명에 있어서 용어, "준성숙 수지상 세포" 는 미성숙 수지상 세포의 특성의 일부를 상실하고, 성숙 수지상 세포의 표현형의 일부 특성을 갖는 수지상 세포를 말한다.In the present invention, the term "semi-mature dendritic cells" refers to dendritic cells that lose some of the characteristics of immature dendritic cells and have some characteristics of the phenotype of mature dendritic cells.

본 발명에 있어서 용어, "면역관용" 이란 특정 항원에 대하여 면역 반응을 나타내지않는 상태를 의미한다. 본 발명에 있어서 "면역관용 수지상 세포 (tolerogenic dendritic cell)" 는 자가 항원에 대한 관용을 유도하고 T 세포의 증식을 억제하는 수지상 세포를 의미한다.In the present invention, the term "immune tolerance" refers to a state that does not exhibit an immune response to a specific antigen. In the present invention, "tolerogenic dendritic cell" refers to a dendritic cell that induces tolerance to an autogenous antigen and inhibits the proliferation of T cells.

본 발명은 내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자에 관한 것이다.The present invention relates to liposome nanoparticles in which an antigen is supported in an inner compartment and an immunotolerant material is supported in a lipid membrane.

상기 면역관용물질은 본 분야에서 사용되는 면역관용물질이라면 제한 없이 사용할 수 있으며, 예를 들어, 라파마이신, 비타민D, 인터루킨-10, 인터루킨-27, 인터루킨-35, 변환성장인자-베타, 덱사메타손(Dexamethasone), 종양괴사인자-알파, 인터페론-알파, 인터페론-람다, 혈관작용장펩티드(vasoactive intestinal peptide; VIP), 글루코코르티코이드(glucocorticoids), 간세포성장인자(hepatocyte growth factor; HGF), 코르티코스테로이드(corticosteroids), 사이클로스포린 A(cyclosporine A), 아드레노메둘린(Adrenomedullin), 아릴 하이드로카본 리셉터(aryl hydrocarbon receptor ligand)(예를 들어, 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester) 포스파티딜세린(phosphatidylserine) 등을 포함할 수 있으며, 예를 들어 라파마이신일 수 있다.The immune tolerant substance can be used without limitation as long as it is an immune tolerant substance used in this field, for example, rapamycin, vitamin D, interleukin-10, interleukin-27, interleukin-35, transformation growth factor-beta, dexamethasone ( Dexamethasone), tumor necrosis factor-alpha, interferon-alpha, interferon-lambda, vasoactive intestinal peptide (VIP), glucocorticoids, hepatocyte growth factor (HGF), corticosteroids ), cyclosporine A, adrenomedullin, aryl hydrocarbon receptor ligand (e.g., 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester ) Phosphatidylserine (phosphatidylserine) may be included, for example, may be rapamycin.

상기 항원은 심근경색이 발병된 개체의 심근경색 부위를 균질화 및 원심분리하여 추출한 용해물 또는 펩티드를 포함할 수 있다. The antigen may include a lysate or peptide extracted by homogenizing and centrifuging a myocardial infarction site of an individual suffering from myocardial infarction.

상기 항원은 자가면역질환을 일으키는 자가면역 항원 또는 항원 대용물일 수 있으며, 예를 들어, RNP/Sm, Sm, SS-A, SS-B, Scl-70, Jo-1, 필라그린, 피브린, 피브리노겐, 비멘틴, 콜라겐, 알파-에놀라제, 글루텔린, 미엘린 염기성 단백질, 아세틸콜린 수용체, 내인성 항원, 미엘린 희소돌기아교세포 당단백질, 췌장 베타-세포 항원, 인슐린, 글루탐산 데카르복실라제 (GAD), II형 콜라겐, 인간연골 gp39, fp130-RAPS, 단백질지질 단백질, 피브릴라린, 소형 핵 단백질, 갑상선 자극 인자 수용체, 히스톤, 당단백질 gp70, 피루베이트 데히드로게나제 데히드로리포아미드 아세틸트랜스퍼라제 (PCD-E2), 모낭 항원, A-글리아덴, 글리아덴, 인슐린, 프로인슐린, 섬-특이적 글루코스-6-포스파타제 촉매 서브유닛-관련 단백질 (IGRP), 인간 트로포미오신 이소형 5, 바히아 그라스(Bahia grass) 꽃가루 (BaGP), 복숭아 알레르겐 Pru p 3, 알파 s1-카세인 밀크 알레르겐, Apig1 셀러리 알레르겐, Bere1 브라질넛 알레르겐, B-락토글로불린 밀크 알레르겐, 소혈청 알부민, Cor a 1.04 헤이즐넛 알레르겐, 미엘린 연관 당단백질, 아쿠아포린, IV형 콜라겐의 α3 사슬, 오브알부민 난 알레르겐, 애드베이트(Advate), 항혈우병 인자, 코지네이트(Kogenate), 엘록테이트(Eloctate), 재조합 인자 VIII Fc 융합 단백질, 리팩토(Refacto), 노보(Novo) VIIa, 재조합 인자 VII, 엡타코그 알파(eptacogalfa), 헬릭세이트(Helixate), 모나닌(Monanine), 응고 인자 IX, 윌레이트(Wilate), 세레다제(Ceredase), 알글루세라제(Alglucerase), 세레자임(Cerezyme), 이미글루세라제(Imiglucerase), 엘렐소(Elelso), 탈리글루세라제 알파(taliglucerase alfa), 파브라자임(Fabrazyme), 아갈시다제(Agalsidase) 베타, 알두자라자임(Aldurazyme), 이두로니다제(iduronidase), 마이오자임(Myozyme), 산-글루코시다제, 엘라프라제(Elaprase), 이두로네이트2-술파타제, 나글라자임(Naglazyme) 아릴술파타제 B, N-아세틸갈락토스아민-4-술파타제 등을 포함할 수 있으나, 이에 제한되지 않는다.The antigen may be an autoimmune antigen or an antigen substitute that causes an autoimmune disease, for example, RNP/Sm, Sm, SS-A, SS-B, Scl-70, Jo-1, filagrin, fibrin, fibrinogen , Vimentin, collagen, alpha-enolase, glutelin, myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic beta-cell antigen, insulin, glutamic acid decarboxylase (GAD), Type II collagen, human cartilage gp39, fp130-RAPS, protein lipoprotein, fibrillarin, small nuclear protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD -E2), hair follicle antigen, A-gliaden, gliaden, insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), human tropomyosin isoform 5, bahia grass ( Bahia grass) Pollen (BaGP), peach allergen Pru p 3, alpha s1-casein milk allergen, Apig1 celery allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 hazelnut allergen, myelin related Protein, aquaporin, α3 chain of type IV collagen, ovalbumin egg allergen, Advate, antihemophilia factor, Kogenate, Eloctate, recombinant factor VIII Fc fusion protein, Refacto ), Novo VIIa, Recombinant Factor VII, Eptacogalfa, Helixate, Monanine, Coagulation Factor IX, Willate, Ceredase, Alglu Alglucerase, Cerezyme, Imiglucerase, Elelso, taliglucerase alfa, Fabrazyme, Agalcidase lsidase) beta, Aldurazyme, iduronidase, myozyme, acid-glucosidase, elaprase, iduronate 2-sulfatase, nagla Naglazyme arylsulfatase B, N-acetylgalactosamine-4-sulfatase, and the like may be included, but are not limited thereto.

상기 리포좀 나노입자는 수화된 지질 필름 과정을 통해 제조될 수 있으며, 예를 들어 지질 유기 용매에 지질을 용해시키는 단계; 상기 지질이 용해된 유기 용매에 면역관용물질을 첨가하는 단계; 상기 유기 용매를 제거하여 지질 필름을 형성하는 단계; 및 상기 지질 필름에 버퍼와 항원을 첨가한 후 수화시키는 단계에 의해 제조될 수 있으나, 이에 제한되지 않는다.The liposome nanoparticles may be prepared through a hydrated lipid film process, for example, dissolving the lipid in a lipid organic solvent; Adding an immunotolerant to the organic solvent in which the lipid is dissolved; Removing the organic solvent to form a lipid film; And hydration after adding a buffer and an antigen to the lipid film, but is not limited thereto.

상기 리포좀 나노입자는 합성된 후 lipid extruder를 이용하여 입자 크기를 조절할 수 있으며, 예를 들어 300 nm 이하의 지름을 갖는 것일 수 있다.After the liposome nanoparticles are synthesized, the particle size may be adjusted using a lipid extruder, and may have, for example, a diameter of 300 nm or less.

상기 면역관용물질은 지질 필름의 형성 단계에서 이중 지질막 내에 담지될 수 있다.The immune tolerant material may be supported in the double lipid membrane in the step of forming the lipid film.

상기 항원은 수화 과정에서 입자에 담지되며, 항원의 음전하와 리포좀의 양전하 간의 정전기적 인력에 의해 담지 효율이 상승할 수 있다.The antigen is carried on the particles during the hydration process, and the carrying efficiency may be increased by an electrostatic attraction between the negative charge of the antigen and the positive charge of the liposome.

상기 이중 지질막은 DOTAP, DOPE, 콜레스테롤 및 DSPE-PEG2000으로 이루어진 군으로부터 선택된 하나 이상의 인지질을 포함할 수 있다.The dual lipid membrane may contain one or more phospholipids selected from the group consisting of DOTAP, DOPE, cholesterol and DSPE-PEG2000.

본 발명의 리포좀 나노입자는 항원 및 면역관용유도물질의 전달체로 사용되는 기존의 PLGA(poly(lactic-co-glycolic acid)) 나노입자와 구조적인 차이가 있으며, 이러한 구조적 차이로 인해 탑재된 단백질의 안정성에 대한 이점을 보인다. 기존의 PLGA 나노입자는 PLGA 폴리머로 구성되어 속이 꽉 차며, 입자 곳곳에 분포하는 미세세공 사이로 단백질을 담지하기 때문에 PLGA 폴리머와 단백질과의 상호작용이 존재한다. PLGA에 존재하는 작용기인 카르복실릭 산(-COOH)과 입자 분해과정에서 생성되는 산성물질은 주변 환경을 국소적으로 높은 산성도를 띠게 하며, 이로 인한 담지된 단백질의 변성이 발생한다. The liposome nanoparticles of the present invention are structurally different from conventional PLGA (poly(lactic-co-glycolic acid)) nanoparticles used as carriers of antigens and immune tolerance inducing substances, and due to these structural differences, the It shows an advantage for stability. Existing PLGA nanoparticles are made of PLGA polymer and are filled with a protein, and the interaction between the PLGA polymer and the protein exists because the protein is carried through the micropores distributed throughout the particle. Carboxylic acid (-COOH), a functional group present in PLGA, and acidic substances produced during particle decomposition locally give the surrounding environment high acidity, resulting in denaturation of the supported protein.

반면, 본 발명의 리포좀 나노입자는 표면은 인지질 이중막으로 구성되어 있으며, 내부는 수분으로 가득 차 있기 때문에, 단백질 탑재 시 PLGA 나노입자에서 나타나는 단백질 변성을 방지할 수 있다. 또한, 합성과정에서 단백질과 유기용매의 혼합이 필요한 PLGA 나노입자와는 달리 리포좀 나노입자는 오직 수용액상에서만 단백질 담지과정이 이루어지므로 PLGA에 비하여 바이오물질을 안정적으로 탑재할 수 있다.On the other hand, since the surface of the liposome nanoparticles of the present invention is composed of a phospholipid double membrane and the inside is filled with moisture, protein denaturation that appears in the PLGA nanoparticles when protein is mounted can be prevented. In addition, unlike PLGA nanoparticles that require mixing of a protein and an organic solvent in the synthesis process, liposome nanoparticles are capable of stably loading biomaterials compared to PLGA because the protein loading process is performed only in aqueous solution.

상기 리포좀 나노입자는 림프절을 표적화하는 것으로, 체내에 투여시 림프절로 이동하여 면역관용 수지상 세포를 성숙시켜 Treg 세포를 활성화시킬 수 있다. 활성화된 Treg 세포는 심장의 면역 반응을 조절하여 심근경색, 뇌졸증 또는 자가면역질환을 치료할 수 있다.The liposomal nanoparticles target lymph nodes, and when administered into the body, they migrate to the lymph nodes and mature dendritic cells for immune tolerance to activate Treg cells. Activated Treg cells can control the immune response of the heart to treat myocardial infarction, stroke or autoimmune disease.

본 발명에서 치료용 조성물은 리포좀 나노입자 외에, 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있으며, 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액 등의 형태로 제형화하여 사용될 수 있다. In the present invention, the therapeutic composition may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions in addition to liposome nanoparticles, and powders, granules, tablets, capsules, suspensions according to conventional methods. , Emulsion, syrup, aerosol, etc. Oral formulations, external preparations, suppositories, or sterile injectable solutions can be formulated and used.

상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.　경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 적어도 한 가지 이상의 부형제 및/또는 윤활제 등을 포함할 수 있다.　경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 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, 비수성 용제, 현탁제, 유제, 동결건조제제, 좌제 등 이 포함될 수 있다.When formulating the composition, it is prepared by using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one or more excipients and/or lubricants. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like.

상기 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 보다 바람직한 효과를 위해서, 본 발명의 조성물의 투여량은 유효성분을 기준으로 1일 0.1 mg/kg 내지 100 mg/kg으로 하는 것이 좋으나 이에 제한되는 것은 아니다.　투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수 있다. 본 발명의 조성물은 동물, 바람직하게는 인간을 포함하는 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 정맥, 근육, 피하주사 등에 의해 투여될 수 있다.The preferred dosage of the composition varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. For a more preferable effect, the dosage of the composition of the present invention is preferably 0.1 mg/kg to 100 mg/kg per day based on the active ingredient, but is not limited thereto. Administration may be administered once a day or may be divided several times. The composition of the present invention can be administered to animals, preferably mammals, including humans, by various routes. All modes of administration can be expected, for example, it can be administered orally, intravenously, intramuscularly, or by subcutaneous injection.

이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by examples. However, these examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

[실시예] [Example]

<제조예 1> <Production Example 1>

도 2에 도시된 바와 같은 공정에 따라 리포좀 나노입자를 합성하였다. 유리용기에 클로로폼에 용해된 인지질을 DOTAP:DOPE:cholesterol:DSPE-PEG2000:rapamycin 의 mol ratio가 0.46:0.35:0.11:0.05 가 되도록 용액을 섞은 후, 공기총 혹은 회전증발농축기를 이용하여 유기용매를 제거하여, 인지질 얇은 막을 유리면에 도포시켰다. 1.21 mM DOTAP, 0.91 mM DOPE, 0.300 mM cholesterol, 0.13 mM DSPE-PEG2000, 0.08 mM rapamycin 이 되도록 심근경색이 유도된 쥐의 심장조직 추출물 항원이 포함된 1x PBS(pH 7.4) 용액으로 인지질을 수화시켰다. 구체적으로, 도포된 인지질의 양이 총 2.62 umol 일 경우, 200 ug 의 항원이 담긴 PBS 용액 1 ml로 인지질을 수화시켰다. 이 후, 1 시간동안 차광조건, 37 ℃, 500 rpm 으로 인지질, 항원 혼합물을 교반시켰다. 수화된 리포좀을 lipid-extruder를 이용하여 멤브레인의 크기 1000, 400, 200 nm 순서대로 반복 사출하였다. 각 사출 과정 시 최소 11 번의 사출을 시행하여 리포좀 나노입자를 합성하였다.Liposome nanoparticles were synthesized according to the process as shown in FIG. 2. The phospholipid dissolved in chloroform is mixed in a glass container so that the mol ratio of DOTAP:DOPE:cholesterol:DSPE-PEG2000:rapamycin is 0.46:0.35:0.11:0.05, and then the organic solvent is mixed with an air gun or a rotary evaporator. After removal, a thin film of phospholipids was applied to the glass surface. Phospholipids were hydrated with 1x PBS (pH 7.4) solution containing the antigen of the heart tissue extract from mice induced myocardial infarction to be 1.21 mM DOTAP, 0.91 mM DOPE, 0.300 mM cholesterol, 0.13 mM DSPE-PEG2000, and 0.08 mM rapamycin. Specifically, when the amount of the applied phospholipid was a total of 2.62 umol, the phospholipid was hydrated with 1 ml of a PBS solution containing 200 ug of antigen. Thereafter, the mixture of phospholipids and antigens was stirred under light blocking conditions at 37° C. and 500 rpm for 1 hour. Hydrated liposomes were repeatedly injected in the order of the membrane size 1000, 400, 200 nm using a lipid-extruder. In each injection process, at least 11 injections were performed to synthesize liposome nanoparticles.

<실시예 1> <Example 1>

제조예 1에서와 같이 합성된 리포좀 나노입자의 입자 크기 및 형태를 확인하고 그 결과를 도 3에 도시하였다. 도 3에서 볼 수 있는 바와 같이, 합성된 리포좀 나노입자는 약 218.0 ± 92.0 nm 이었으며, TEM 이미지를 통해 구형임을 확인하였다. 음 전하를 띠는 항원단백질을 담지함에 따라 양 전하를 띠는 blank 리포좀에 비교하여 전하가 감소하였다.The particle size and shape of the liposome nanoparticles synthesized as in Preparation Example 1 were confirmed, and the results are shown in FIG. 3. As can be seen in FIG. 3, the synthesized liposome nanoparticles were about 218.0 ± 92.0 nm, and it was confirmed that they are spherical through TEM images. As the negatively charged antigenic protein was loaded, the charge decreased compared to the positively charged blank liposome.

리포좀을 1% Triton-X 용액에 용해시켜, 담지된 항원의 양을 BCA 테스트를 통해 확인하였으며, 탑재된 라파마이신의 양은 UV-Vis 스펙트럼 분석기를 이용하여 검출하였다. 항원 혹은 라파마이신만을 리포좀에 탑재할 때에 비하여 두 가지 모두 탑재한 경우, 서로 경쟁적 반응을 하여 한 가지만 탑재할 때보다 담지 효율이 감소하는 것을 확인하였다. 리포좀에 담지되는 항원과 라파마이신의 양을 확인하고 그 결과를 표 1에 나타냈다.The liposome was dissolved in 1% Triton-X solution, and the amount of supported antigen was confirmed through BCA test, and the amount of mounted rapamycin was detected using a UV-Vis spectrum analyzer. When both antigens or rapamycin were loaded on the liposome, it was confirmed that when both were loaded, they reacted competitively with each other and the loading efficiency decreased compared to when only one was loaded. The amount of antigen and rapamycin carried on the liposome was confirmed, and the results are shown in Table 1.

<실시예 2><Example 2>

제조예 1에서와 같이 리포좀 나노입자를 합성하되, 리포좀의 이중막에 DiI dye를 삽입하고, 항원에 FITC 를 접합시킨 후 입자를 합성하였다. 도 4a에서 볼 수 있는 바와 같이, DiI와 FITC 형광이 colocalization 되어있는 것을 통해 항원이 입자에 담지된 것을 확인할 수 있었다.The liposome nanoparticles were synthesized as in Preparation Example 1, but DiI dye was inserted into the double membrane of the liposome, and the particles were synthesized after conjugating FITC to the antigen. As can be seen in Figure 4a, it was confirmed that the antigen was carried on the particles through the colocalization of DiI and FITC fluorescence.

또한, 라파마이신의 담지를 확인하기 위해 DSC 열분석을 실시하였다.In addition, DSC thermal analysis was performed to confirm the support of rapamycin.

라파마이신은 소수성을 지니며 수용액에서 크리스탈 형태로 존재한다. 도 4b에서 볼 수 있는 바와 같이, 수용액 내의 라파마이신은 크리스탈 형태로 존재하는 반면, 리포좀 나노입자에 담지된 후에는 수용액 내에 크리스탈이 발견되지 않고 리포좀 나노입자만 관찰되었다. DSC 열분석 결과는 도 4c에 도시하였으며, 순수 라파마이신과는 달리 리포좀에 담지된 라파마이신은 수용액 상에서 크리스탈에 의한 열 반응이 관찰되지 않음을 확인하였다. 이는 리포좀에 담지된 라파마이신은 크리스탈 형태로 존재하기 보다 리포좀 내에서 분자로 존재함을 의미한다. 또한, 도 4d에 서 볼 수 있는 바와 같이, UV 를 통해 리포좀에 라파마이신이 존재하는 것을 확인할 수 있었다. Rapamycin is hydrophobic and exists in crystalline form in aqueous solutions. As can be seen in FIG. 4B, while rapamycin in the aqueous solution exists in a crystal form, after being supported on the liposome nanoparticles, no crystals were found in the aqueous solution, and only liposome nanoparticles were observed. The DSC thermal analysis results are shown in FIG. 4C, and it was confirmed that unlike pure rapamycin, the rapamycin supported on the liposome did not observe a thermal reaction by crystals in an aqueous solution. This means that the rapamycin carried on the liposome exists as a molecule in the liposome rather than in a crystal form. In addition, as can be seen in Figure 4d, it was confirmed that the presence of rapamycin in the liposome through UV.

<실시예 3><Example 3>

제조예 1에서와 같이 리포좀 나노입자를 합성하되, 리포좀의 이중막에 DiI dye를 삽입하고, 수지상 세포에 처리하여 입자의 세포로의 uptake 수준을 관찰하였다. 도 5에서 볼 수 있는 바와 같이, 수지상 세포의 DAPI로 염색된 핵 인근에 붉은 색 형광을 띠는 리포좀 나노입자가 축적되어 있음을 형광현미경으로 관찰할 수 있으며, 유세포 분석기를 통해 약 90 %의 수지상 세포가 입자를 uptake 하였음을 확인할 수 있었다.The liposome nanoparticles were synthesized as in Preparation Example 1, but DiI dye was inserted into the double membrane of the liposome, and the dendritic cells were treated to observe the level of uptake of the particles to the cells. As can be seen in FIG. 5, it can be observed with a fluorescence microscope that red fluorescent liposome nanoparticles are accumulated near the nuclei stained with DAPI of dendritic cells, and about 90% of dendritic cells through flow cytometry It was confirmed that the cells uptake the particles.

<실시예 4><Example 4>

본 발명의 리포좀 나노입자의 항원 전달 능력을 증명하기 위해 유세포 분석기 및 공초점 현미경을 이용한 분석을 실시하였다. 군은 순수한 항원-FITC 및 라파마이신 처리가 된 수지상 세포군과 항원-FITC 및 라파마이신이 탑재된 리포좀이 처리된 수지상 세포군으로 설정되었다. 형광물질 FITC 를 항원에 EDC/NHS 반응을 통하여 연결시킨 후 제조예 1에서와 같이 리포좀 나노입자를 합성하였다. FITC의 양은 UV-Vis 분광기를 통해 정량한 후 각 군에 동일 양의 항원-FITC를 처리한다. 도 6a에서 볼 수 있는 바와 같이 수지상 세포의 항원 uptake는 입자에 탑재시켜 전달한 경우가 순수한 항원을 전달할 때에 비해 우수했으며(6.2% vs 94.9%), MFI 값을 통해 정량한 결과 통계적 유의성이 있음을 확인할 수 있었다.Analysis was performed using a flow cytometer and a confocal microscope in order to prove the antigen delivery ability of the liposome nanoparticles of the present invention. The group was set as a pure antigen-FITC and rapamycin-treated dendritic cell group and antigen-FITC and rapamycin-loaded liposome-treated dendritic cell group. After linking the fluorescent substance FITC to the antigen through EDC/NHS reaction, liposome nanoparticles were synthesized as in Preparation Example 1. The amount of FITC was quantified through UV-Vis spectroscopy, and then the same amount of antigen-FITC was treated in each group. As can be seen in Figure 6a, the antigen uptake of dendritic cells was superior to the case of delivering pure antigen (6.2% vs 94.9%) when delivered by mounting on particles, and as a result of quantification through MFI value, it was confirmed that there was statistical significance. Could

도 6a와 같이 본 발명의 리포좀은 항원 전달 능력이 우수하여 순수한 항원만 전달하였을 때보다 수지상 세포에 강하고 지속적인 항원표출이 가능하다. 또한 도 6b에서 볼 수 있는 바와 같이 입자로 항원 전달 시 24~48 시간까지 lysosome에 남아 항원 분해과정이 진행되며, 세포표면에는 순수 항원만 전달했을 때보다 강한 항원-FITC 시그널이 발생했다. 이는 본 발명의 리포좀 나노입자가 항원 전달에 우수하며, 수지상 세포가 효율적으로 항원제시하는 것을 가능하게 한다는 것을 보여준다.As shown in FIG. 6A, the liposome of the present invention has excellent antigen delivery ability, and thus it is possible to express stronger and sustained antigen to dendritic cells than when only pure antigen is delivered. In addition, as can be seen in Figure 6b, when the antigen is delivered to the particles, the antigen decomposition process remains in the lysosome until 24 to 48 hours, and a stronger antigen-FITC signal was generated on the cell surface than when only pure antigen was delivered. This shows that the liposome nanoparticles of the present invention are excellent in antigen delivery, and dendritic cells can efficiently present antigens.

<실시예 5><Example 5>

제조예 1에서와 같이 합성된 리포좀 나노입자의 면역관용 수지상 세포 유도 능력을 검증하였다. 실험군은 아무 처리되지 않은 수지상 세포 즉, 미성숙 수지상 세포(im), 항원이 담지된 리포좀 나노입자가 처리된 수지상 세포(L-Ag), 항원과 라파마이신이 함께 탑재된 리포좀 나노입자가 처리된 수지상 세포(L-Ag/R), 성숙 수지상 세포(m)로 구성되며, 면역관용 수지상 세포 및 성숙 수지상 세포의 분화에 필요되는 어쥬번트로 LPS가 사용되었다. 수지상 세포표면에 존재하는 공동자극분자인 CD40, CD80, CD86과 MHC II의 발현량을 유세포 분석기를 통해 관찰 및 정량하였으며, 도 7a에서 볼 수 있는 바와 같이 항원 및 라파마이신이 탑재된 리포좀이 처리된 수지상 세포군에서 CD40, CD80, CD86의 발현량이 성숙 수지상 세포에 비해 감소하였으며, MFI 값 정량을 통해 통계적 유의성을 확인하였다. The ability of the liposome nanoparticles synthesized as in Preparation Example 1 to induce immune-tolerant dendritic cells was verified. The experimental group consisted of untreated dendritic cells, i.e. immature dendritic cells (im), dendritic cells treated with antigen-loaded liposomal nanoparticles (L-Ag), and dendritic cells treated with liposomal nanoparticles loaded with antigen and rapamycin. Consisting of cells (L-Ag/R) and mature dendritic cells (m), LPS was used as an adjuvant required for differentiation of immunotolerant dendritic cells and mature dendritic cells. The expression levels of CD40, CD80, CD86 and MHC II, which are co-stimulatory molecules present on the dendritic cell surface, were observed and quantified through flow cytometry, and as shown in FIG. 7A, liposomes loaded with antigen and rapamycin were treated. In the dendritic cell group, the expression levels of CD40, CD80, and CD86 were decreased compared to mature dendritic cells, and statistical significance was confirmed through quantification of MFI values.

분화유도된 면역관용 수지상 세포의 mRNA 수준 변화를 중합효소연쇄반응을 통해 확인하였으며 이 결과를 7b에 도시하였다. 항원과 라파마이신이 동시에 탑재된 나노입자가 처리된 수지상 세포군에서 면역관용 수지상 세포의 특징인 IDO mRNA 수준이 다른 군에 비해 증가하였으며, 라파마이신의 작용효과로 인해 수지상 세포의 이동능력을 높여주는 CCR7 mRNA 수준도 증가하였다. 또한, 염증성 사이토카인인 종양괴사인자-알파, 인터페론-감마의 mRNA은 감소하였으며, 항염증성 사이토카인인 변환성장인자-베타의 mRNA 수준은 증가하였다.The change in mRNA level of the differentiation-induced immunotolerant dendritic cells was confirmed through polymerase chain reaction, and the result is shown in 7b. In the dendritic cell group treated with nanoparticles loaded with antigen and rapamycin at the same time, the level of IDO mRNA, a characteristic of immune-tolerant dendritic cells, increased compared to other groups, and CCR7, which enhances the ability of dendritic cells to move due to the action effect of rapamycin mRNA levels also increased. In addition, mRNA levels of inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, decreased, and mRNA levels of anti-inflammatory cytokine transforming growth factor-beta were increased.

본 발명의 리포좀 나노입자에 의해 유도된 면역관용 수지상 세포의 T 세포 증식 억제능력을 CFSE 희석 에세이를 통해 검증하였다. 군은 도 7a 실험과 동일하며, 면역관용 혹은 성숙 수지상 세포를 유도한 후 CD4 T 세포와 공배양하여 5일 후 CFSE 에세이를 유세포 분석기를 통해 시행하였다. 도 7c에서 볼 수 있는 바와 같이 입자에 의해 유도된 면역관용 수지상세포(tDC)는 성숙 수지상 세포(mDC)에 비해 CFSE^dim에 속하는 CD4 T 세포 비율이 낮으며 미성숙 수지상 세포(imDC)군과 비슷한 수준으로 검출되었다. The ability of the immunogenic dendritic cells to inhibit T cell proliferation induced by the liposomal nanoparticles of the present invention was verified through a CFSE dilution assay. The group was the same as that of the experiment of Fig. 7a, and after 5 days of co-culture with CD4 T cells after inducing immune-tolerant or mature dendritic cells, CFSE assay was performed through flow cytometry. As can be seen in Figure 7c, the immunotolerant dendritic cells (tDC) induced by the particles have a lower proportion of CD4 T cells belonging to CFSE ^dim compared to the mature dendritic cells (mDC) and are similar to the immature dendritic cells (imDC) group. Was detected.

간략하게, 수지상 세포가 본 발명의 리포좀 입자에 의하여 면역관용 수지상 세포로 분화되었음을 표면인자, 유전자 변화 수준, T세포 증식 억제능력 분석을 통하여 검증하였다.Briefly, it was verified that dendritic cells were differentiated into immune-tolerant dendritic cells by the liposome particles of the present invention through analysis of surface factors, gene change levels, and T cell proliferation inhibitory ability.

<실시예 6><Example 6>

제조예 1에서와 같이 합성된 리포좀 나노입자를 수지상 세포에 처리하여 실시예 5와 같이 면역관용 수지상 세포 유도를 유도한 후, 비장 세포와 공배양을 통하여 조절 T 세포 분화를 유도하였다. 군은 미성숙 수지상 세포와 공배양된 세포(im), 항원을 탑재한 입자와 수지상 세포와 공배양된 세포(L-Ag), 항원 및 라파마이신을 담지한 수지상 세포와 공배양된 세포(L-Ag/R), 성숙 수지상 세포와 공배양된 세포(m)수지상 세포로 설정되었다. 비장 세포는 1:10의 비율로 공배양되었으며, 배양 후 72 시간 뒤 유세포 분석기를 통하여 CD3, CD4 인자로 게이팅 후 조절 T 세포 마커 인자인 Foxp3 함량을 분석하였다. 도 8a에서 볼 수 있는 바와 같이 다른 군에 비교하여 L-Ag/R을 처리하여 유도된 면역관용 수지상 세포는 Foxp3+ T 세포의 비율이 높았으며, CD3+CD4+ T 세포중에서 Foxp3+ T 세포은 약 1.6 % 인 것으로 확인할 수 있었다. 해당 경향은 mRNA 수준에서도 유사하였으며, 미성숙 수지상 세포에 비해 3배가량 높은 Foxp3 mRNA가 검출되었다.The liposomal nanoparticles synthesized as in Preparation Example 1 were treated on dendritic cells to induce immune-tolerant dendritic cell induction as in Example 5, and then regulatory T cell differentiation was induced through co-culture with spleen cells. In the group, cells co-cultured with immature dendritic cells (im), cells co-cultured with antigen-bearing particles and dendritic cells (L-Ag), and cells co-cultured with dendritic cells carrying antigen and rapamycin (L- Ag/R), cells co-cultured with mature dendritic cells (m) dendritic cells. Splenocytes were co-cultured at a ratio of 1:10, and after 72 hours after culture, the content of Foxp3, a regulatory T cell marker factor, was analyzed after gating with CD3 and CD4 factors through flow cytometry. As can be seen in Figure 8a, compared to the other groups, the immunotolerant dendritic cells induced by treatment with L-Ag/R had a high proportion of Foxp3+ T cells, and among CD3+CD4+ T cells, Foxp3+ T cells were about 1.6%. It could be confirmed. This trend was similar at the mRNA level, and Foxp3 mRNA was detected three times higher than that of immature dendritic cells.

리포좀 나노입자 및 면역관용 수지상 세포에 의하여 유도된 조절 T 세포의 기능을 확인하기 위해 중합효소 연쇄반응을 실시하였다. 염증성 사이토카인인 종양괴사인자-알파와 인터페론-감마의 mRNA 검출량은 성숙 수지상 세포와 공배양된 세포군에 비해 30 % 수준으로 감소하였으며, 항염증성 사이토카인인 인터루킨-10 mRNA 검출량은 미성숙 수지상 세포와 공배양된 세포군에 비해 약 8 배가량 증가하였다.Polymerase chain reaction was performed to confirm the function of regulatory T cells induced by liposome nanoparticles and immune tolerant dendritic cells. The amount of mRNA detected for tumor necrosis factor-alpha and interferon-gamma, which are inflammatory cytokines, decreased to 30% compared to the cell group co-cultured with mature dendritic cells, and the amount of detection of interleukin-10 mRNA, an anti-inflammatory cytokine, was similar to that of immature dendritic cells. It increased about 8 times compared to the cultured cell group.

<실시예 7><Example 7>

리포좀 나노입자가 림프절을 표적화하는지를 확인하기 위해, 형광물질 FITC 를 항원에 EDC/NHS 반응을 통하여 연결시킨 후 리포좀 나노입자에 담지시켰다. 2 mg/kg LPS를 마우스에 복강주사하여 체내 염증을 유도한다. 주사한 후 24 시간뒤에, FITC 가 연결된 항원과 라파마이신을 함유한 리포좀 나노입자를 피하주사하며 형광의 세기를 24 시간 후에 확인하였다. In order to confirm whether the liposomal nanoparticles target lymph nodes, the fluorescent substance FITC was ligated to the antigen through EDC/NHS reaction, and then loaded on the liposome nanoparticles. Intraperitoneal injection of 2 mg/kg LPS into mice induces inflammation in the body. 24 hours after injection, liposome nanoparticles containing FITC-linked antigen and rapamycin were injected subcutaneously, and the intensity of fluorescence was checked after 24 hours.

도 9에서 볼 수 있는 바와 같이, 항원이 리포좀 나노입자를 통하여 림프절로 전달된 것을 확인할 수 있었다.As can be seen in Figure 9, it was confirmed that the antigen was delivered to the lymph nodes through the liposome nanoparticles.

<실시예 8><Example 8>

도 10에 도시된 실험 디자인에 따라 리포좀 나노입자에 의한 심근경색 치료 효과를 검증하였다.According to the experimental design shown in FIG. 10, the effect of treating myocardial infarction by liposome nanoparticles was verified.

우선, 급성 심근경색 마우스 모델을 제작하였다. 5주령 된 C57BL/6 마우스(수컷)를 중앙실험동물(한국)에서 구입하여, 온도 23℃, 습도 55%, 조도 300-500 lux, 명암주기 12 시간의 사육환경이 유지된 사육장에서 사육하였다. 사료는 ㈜ 퓨리나의 실험동물 멸균고형사료(Purina Rodent Laboratory Chow 5001, Purina Mills, USA)를 구입하여 자유로이 공급하였으며 음용수는 멸균증류수을 자유롭게 섭취할 수 있도록 하였다. 2 주간의 순화과정을 거친 후, 마취된 마우스에 22G angio needle을 이용하여 기관내삽관(intubation)하고, 마우스 흉부의 털을 제거한 뒤 피부를 절개하여 갈비뼈를 살짝 벌리고 심장의 심막(pericardium)을 제거하였다. 그 후 관상동맥(cordonary artery)의 좌전하행지(left anterior descending artery)를 나일론 실크(nylon silk)로 살짝 떠서 완전히 결찰시켰다. 이후 마우스 폐의 공기를 빼고 수술부위를 봉합하였다.First, a mouse model of acute myocardial infarction was produced. Five-week-old C57BL/6 mice (males) were purchased from Central Experimental Animals (Korea), and reared in a breeding facility maintained at a temperature of 23°C, humidity 55%, illuminance 300-500 lux, and a 12-hour light/dark cycle. For feed, Purina Rodent Laboratory Chow 5001 (Purina Mills, USA) was purchased and supplied freely, and sterilized distilled water was freely available for drinking. After 2 weeks of acclimatization, the anesthetized mouse was intubated using a 22G angio needle, and the hairs on the chest of the mouse were removed, and then the skin was incised to slightly open the ribs and remove the pericardium of the heart. I did. After that, the left anterior descending artery of the coronary artery was slightly scooped with nylon silk and completely ligated. After that, air was removed from the mouse lung and the surgical site was sutured.

그런 다음 제조예 1에서와 같이 제조된 항원과 라파마이신이 담지된 리포좀 나노입자(L-Ag/R)를 마우스에 피하 주사한 후 4주 동안 심초음파 검사법을 통해 심장의 수축기말 부피(ESV)와 박출률(EF)를 측정하여 심장기능을 분석하였다. 또한 심근 경색의 중증도를 밝히기 위해 각각의 심장으로부터 얻은 섹션에 MT stain을 실시하였다. 경색 면적 및 비-경색 면적의 측정을 통해 경색 크기를 결정하였다. 상대적인 경색 크기를 100%로 추정하였고 [경색 면적/(경색 면적 + 비-경색 면적)]으로 계산하였다.Then, after subcutaneous injection of liposomal nanoparticles (L-Ag/R) loaded with antigen and rapamycin prepared as in Preparation Example 1 into a mouse, the end-systolic volume (ESV) of the heart through echocardiography for 4 weeks. Cardiac function was analyzed by measuring and ejection rate (EF). In addition, MT staining was performed on sections obtained from each heart to determine the severity of myocardial infarction. Infarct size was determined through measurement of infarct area and non-infarct area. The relative infarct size was estimated as 100% and calculated as [infarct area/(infarct area + non-infarct area)].

대조군으로서 항원과 라파마이신이 담지되지 않은 리포좀 나노입자(Blank)를 사용하였다.As a control, liposome nanoparticles (Blank) not loaded with antigen and rapamycin were used.

도 11a에서 볼 수 있는 바와 같이, 심초음파를 통해서 심장기능을 확인한 결과 L-Ag/R 주입군이 blank 입자 주입군에 비해서 end-systolic volumes (ESV)가 감소하고 Ejection fraction(EF)가 증가하는 것을 확인할 수 있었으며, 이는 L-Ag/R에 의해 심근경색 후의 심장 기능이 회복됨을 보여주는 것이다.As can be seen in FIG. 11A, as a result of confirming cardiac function through echocardiography, the end-systolic volumes (ESV) of the L-Ag/R injection group decrease and the ejection fraction (EF) increases compared to the blank particle injection group. It was confirmed that the cardiac function after myocardial infarction is restored by L-Ag/R.

또한, 도 11b에서 볼 수 있는 바와 같이, 심근경색 4주 후에 마우스의 심장 조직을 MT stain해서 경색 크기를 비교하였을 때, blank 입자군에 비해서 L-Ag/R을 주입한 마우스군의 심장에서 경색 크기가 감소하였고 collagen area 도 감소하는 것을 확인하였다. 이는 L-Ag/R에 의해서 심근경색후 remodeling을 감소시켰음을 보여주는 결과이다.In addition, as can be seen in Figure 11b, when comparing the infarct size by MT staining the heart tissue of the mouse 4 weeks after myocardial infarction, infarction in the heart of the mouse group injected with L-Ag/R compared to the blank particle group It was confirmed that the size decreased and the collagen area also decreased. This is a result showing that L-Ag/R reduced remodeling after myocardial infarction.

<실시예 9><Example 9>

심근경색 조직에서 Ag-NP에 의한 염증 억제 효과를 확인하기 위하여 심근경색 1주 후에 마우스의 심장에서 염증성 대식세포와 회복성 대식세포의 분포를 면역형광항체법을 사용하여 확인하였다. 도 12에서 볼 수 있는 바와 같이 염증성 대식세포의 마커인 CD68과 iNOS를 동시염색 했을 경우 blank 입자에 비해서 L-Ag/R 입자를 주입한 마우스의 심장에서 염증성 대식세포가 감소하였음을 확인하였다. 반면에 회복성 대식세포의 마커인 CD68과 MR을 동시염색 했을 경우 blank 입자에 비해서 L-Ag/R를 주입한 마우스의 심장에서 회복성 대식세포가 증가하였음을 확인하였다. 이는 L-Ag/R에 의해서 심근경색 후의 염증성 대식세포가 감소하고 회복성 대식세포가 증가됨으로써 심근경색후 면역반응을 조절하고 있음을 보여주는 결과이다. In order to confirm the inhibitory effect of Ag-NP on inflammation in myocardial infarction tissue, the distribution of inflammatory macrophages and restorative macrophages in the heart of mice 1 week after myocardial infarction was confirmed using the immunofluorescent antibody method. As can be seen in FIG. 12, when simultaneously staining CD68 and iNOS, markers of inflammatory macrophages, it was confirmed that inflammatory macrophages were reduced in the heart of mice injected with L-Ag/R particles compared to blank particles. On the other hand, when the markers of recoverable macrophages, CD68 and MR were co-stained, it was confirmed that the number of recoverable macrophages increased in the heart of mice injected with L-Ag/R compared to the blank particles. This is a result showing that L-Ag/R regulates the immune response after myocardial infarction by decreasing inflammatory macrophages after myocardial infarction and increasing recoverable macrophages.

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (11)

내부 구획에 항원이 담지되고 이중 지질막 내에 면역관용물질이 담지된 리포좀 나노입자. Liposomal nanoparticles carrying antigens in the inner compartment and immune tolerant substances in the lipid membrane. 제1항에 있어서,
상기 면역관용물질은 라파마이신, 비타민D, 인터루킨-10, 인터루킨-27, 인터루킨-35, 변환성장인자-베타, 덱사메타손(Dexamethasone), 종양괴사인자-알파, 인터페론-알파, 인터페론-람다, 혈관작용장펩티드(vasoactive intestinal peptide; VIP), 글루코코르티코이드(glucocorticoids), 간세포성장인자(hepatocyte growth factor; HGF), 코르티코스테로이드(corticosteroids), 사이클로스포린 A(cyclosporine A), 아드레노메둘린(Adrenomedullin), 아릴 하이드로카본 리셉터(aryl hydrocarbon receptor ligand) 및 포스파티딜세린(phosphatidylserine)으로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것인 리포좀 나노입자.The method of claim 1,
The immune tolerant substances are rapamycin, vitamin D, interleukin-10, interleukin-27, interleukin-35, transformation growth factor-beta, dexamethasone, tumor necrosis factor-alpha, interferon-alpha, interferon-lambda, vascular action Vasoactive intestinal peptide (VIP), glucocorticoids, hepatocyte growth factor (HGF), corticosteroids, cyclosporine A, adrenomedullin, aryl hydro Liposome nanoparticles comprising at least one selected from the group consisting of carbon receptors (aryl hydrocarbon receptor ligand) and phosphatidylserine (phosphatidylserine). 제1항에 있어서,
상기 항원은 심근경색이 발병된 개체의 심근경색 부위를 균질화 및 원심분리하여 추출한 용해물 또는 펩티드를 포함하는 것인 리포좀 나노입자.The method of claim 1,
The antigen is a liposome nanoparticle comprising a lysate or peptide extracted by homogenizing and centrifuging the myocardial infarction site of an individual suffering from myocardial infarction. 제1항에 있어서,
상기 항원은 자가면역 항원을 포함하는 것인 리포좀 나노입자.The method of claim 1,
The antigen is liposome nanoparticles comprising an autoimmune antigen. 제1항에 있어서,
상기 리포좀 나노입자는 300 nm 이하의 지름인 것인 리포좀 나노입자.The method of claim 1,
The liposome nanoparticles are liposome nanoparticles having a diameter of 300 nm or less. 제1항에 있어서,
상기 리포좀 나노입자는 하기 단계에 의해 제조되는 것인 리포좀 나노입자:
유기 용매에 지질을 용해시키는 단계;
상기 지질이 용해된 유기 용매에 면역관용물질을 첨가하는 단계;
상기 유기 용매를 제거하여 지질 필름을 형성하는 단계; 및
상기 지질 필름에 버퍼와 항원을 첨가한 후 수화시키는 단계.The method of claim 1,
The liposome nanoparticles are prepared by the following steps:
Dissolving the lipid in an organic solvent;
Adding an immunotolerant to the organic solvent in which the lipid is dissolved;
Removing the organic solvent to form a lipid film; And
Hydrating after adding a buffer and an antigen to the lipid film. 제6항에 있어서,
상기 면역관용물질은 지질 필름의 형성 단계에서 이중 지질막 내에 담지되는 것인 리포좀 나노입자. The method of claim 6,
The immunotolerant material is a liposome nanoparticle that is supported in a double lipid membrane in the step of forming a lipid film. 제6항에 있어서,
상기 이중 지질막은 DOTAP, DOPE, 콜레스테롤 및 DSPE-PEG2000으로 이루어진 군으로부터 선택된 하나 이상의 인지질을 포함하는 것인 리포좀 나노입자.The method of claim 6,
The dual lipid membrane is a liposome nanoparticle comprising at least one phospholipid selected from the group consisting of DOTAP, DOPE, cholesterol and DSPE-PEG2000. 제1항 내지 제8항 중 어느 한 항의 리포좀 나노입자를 포함하는 크론병, 루푸스, 제1당뇨병, 류마티스 관절염, 이식편대숙주질환(GvHD), 알러지, 천식, 간염, 궤양성 대장염, 혈우병, 골 관절염, 척수손상질환, 뇌졸중 또는 심근경색 치료용 조성물. Crohn's disease, lupus, first diabetes, rheumatoid arthritis, graft versus host disease (GvHD), allergy, asthma, hepatitis, ulcerative colitis, hemophilia, bone containing the liposomal nanoparticles of any one of claims 1 to 8 Composition for the treatment of arthritis, spinal cord injury, stroke or myocardial infarction. 제9항에 있어서,
상기 리포좀 나노입자가 림프절을 표적화하는 것인 조성물.The method of claim 9,
The composition wherein the liposome nanoparticles target lymph nodes. 제9항에 있어서,
상기 리포좀 나노입자가 면역관용 수지상 세포를 성숙시켜 Treg 세포를 활성화시키는 것인 조성물.The method of claim 9,
The composition of the liposome nanoparticles to activate Treg cells by maturing immune-tolerant dendritic cells.

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