KR20180015807A - Complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs, method for preparing the same and transporter carrying and releasing drugs using the complex nanoparticle - Google Patents

Complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs, method for preparing the same and transporter carrying and releasing drugs using the complex nanoparticle Download PDF

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KR20180015807A
KR20180015807A KR1020160099228A KR20160099228A KR20180015807A KR 20180015807 A KR20180015807 A KR 20180015807A KR 1020160099228 A KR1020160099228 A KR 1020160099228A KR 20160099228 A KR20160099228 A KR 20160099228A KR 20180015807 A KR20180015807 A KR 20180015807A
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keratin
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pharmaceutical composition
liver
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황유식
박기동
이상천
이재형
이윤기
박나정
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경희대학교 산학협력단
아주대학교산학협력단
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Abstract

The present invention relates to pharmaceutical compositions for liver protection comprising keratin, PEGylated keratin or PEGylated keratin nanoparticles and pharmaceutical compositions for prevention or treatment of hepatotoxic diseases. The keratin, PEGylated keratin or PEGylated keratin nanoparticles have low toxicity or no toxicity, significantly elevate the expression of UGT1A1 and related receptors, and have hepatocyte protective action and hepatotoxicity inhibiting action, thereby being usefully used as a composition for liver protection or a composition for prevention or treatment of hepatotoxic diseases.

Description

ＰＥＧｙｌａｔｅｄ 케라틴을 포함하는 약제의 부작용 완화 기능을 탑재한 복합 나노입자, 이의 제조방법 및 상기 나노입자를 이용한 약제의 담지 및 방출 수송체{Complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs, method for preparing the same and transporter carrying and releasing drugs using the complex nanoparticle}The present invention relates to a composite nanoparticle having a side effect relieving function of a drug including PEGylated keratin, a method for preparing the same, and a method for preparing a nanoparticle containing a complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs same and transporter carrying and releasing drugs using the complex nanoparticle}

본 발명은 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자를 포함하는 간 보호용 약학적 조성물 및 간독성 질환 예방 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to pharmaceutical compositions for liver protection comprising keratin, PEGylated keratin or PEGylated keratin nanoparticles and pharmaceutical compositions for the prevention or treatment of hepatotoxic diseases.

간은 인간의 신체 장기 중 생체 내 대사가 가장 활발하게 일어나는 장기로 인체 내 소화기계와 전신순환계 사이에 위치하면서 외부에서 들어온 생체 외 물질로부터 전신을 방어하는 기능을 수행한다. 생체 내로 들어온 생체 외 물질은 일단 간을 통과하게 되므로 간은 독성물질에 노출될 위험이 다른 장기보다 많고 그만큼 손상될 확률도 매우 높다. 간은 재생능력이 비교적 우수한 장기이지만, 손상이 지속될 경우에는 간 조직의 일부가 완전히 파괴되고 간 기능도 저하되는 등 정상 간으로의 회복이 어려운 상태가 된다. 간질환은 병이 생기는 원인에 따라 바이러스성 간질환, 알코올성 간질환, 약물성 간질환, 지방간, 자가 면역성 간질환, 대사성 간질환 및 기타 간질환으로 구분된다. 간 질환은 초기에 자각증상이 없어 상당히 진행되어서야 발견되기 때문에, 우리나라뿐만 아니라 세계적으로도 사망원인의 수위를 차지하고 있으나, 효과적인 치료제 및 진단방법이 없는 실정이다. 종래 간질환 치료제로서 간 기능 보조제, 항바이러스제, 간세포 촉진제, 면역억제제, 섬유화억제제. 인터페론 등이 사용되고 있지만, 위와 같은 치료제들은 간질환에 효과적이지 못하고 부작용 및 재발의 위험이 높기 때문에 보다 효과적으로 간 세포를 보호하고, 간독성 억제활성을 가진 물질 탐색이 이루어지고 있다.The liver is the organ in which the metabolism of the body is most active in the human body. It is located between the internal digestive system and the systemic circulatory system and functions to defend the whole body from the externally introduced substance. Since the in vivo substance entering the living body passes through the liver, the liver is more likely to be exposed to the toxic substance than the other organs, and the probability of the damage is also very high. The liver is a relatively excellent organ for regeneration. However, if the damage continues, part of the liver tissue is completely destroyed and the liver function is also lowered, making it difficult to recover to normal liver. Liver disease is classified into viral liver disease, alcoholic liver disease, drug substance liver disease, fatty liver, autoimmune liver disease, metabolic liver disease and other liver diseases depending on the cause of disease. Since liver disease is not recognized until it has progressed considerably since it has no subjective symptoms at the beginning, it is the cause of death not only in Korea but also in the world. However, there is no effective treatment and diagnosis method. Conventional liver disease therapeutic agents include liver function supplements, antiviral agents, hepatocyte promoters, immunosuppressants, fibrosis inhibitors. Interferon, etc. However, the above therapeutic agents are not effective for liver disease and have a high risk of side effects and recurrence, so that they are more effective in protecting liver cells and searching for substances having hepatotoxicity-inhibiting activity.

한편, 케라틴은 모발, 손톱, 발톱, 깃털 등을 구성하는 구조 단백질이다. 일반적으로 케라틴은 히스티딘 : 리신 : 아르기닌이 약 1 : 4 : 12의 몰비로 이루어져 있으며, 시스틴의 함량이 많아 황의 함량이 3 내지 5 %를 차지한다. 케라틴은 용해도가 낮아 보통의 용매에는 거의 녹지 않는다. 케라틴은 생체에 포함되어 있는 물질이기 때문에, 생체적합성이 매우 높다. On the other hand, keratin is a structural protein constituting hair, nails, claws, feathers and the like. Generally, keratin is composed of histidine: lysine: arginine in a molar ratio of about 1: 4: 12, and the cystine content is high, so that the content of sulfur is 3 to 5%. Keratin is low in solubility and almost insoluble in common solvents. Since keratin is a substance contained in living bodies, it is highly biocompatible.

다른 한편, 폴리에틸렌글리콜(polyethylene glycol, 이하 PEG)은 HO-(-CH2CH2O-)n-H의 구조를 갖는 고분자 화합물로, 친수성이 강하기 때문에 다양한 단백질에 결합시켜 그 용해도를 증가시킬 수 있다. 단백질에 결합 가능한 PEG의 분자량 범위는 대략 1,000-100,000으로, PEG 분자량이 1,000 이상일 경우에는 독성이 상당히 낮은 편으로 알려져 있다. On the other hand, polyethylene glycol (hereinafter referred to as PEG) is a polymer compound having a structure of HO - (- CH 2 CH 2 O-) n -H. Since it is highly hydrophilic, it can bind to various proteins and increase its solubility have. The molecular weight range of PEG capable of binding to the protein is approximately 1,000-100,000, and when the PEG molecular weight is 1,000 or more, it is known that the toxicity is extremely low.

그런데, 종래 케라틴 또는 PEG화된 케라틴이 간 보호 활성이 있는지에 대해서는 전혀 알려진 바 없다. However, it is not known at present whether keratin or PEGylated keratin has liver protecting activity.

Journal of Drug Delivery Volume 2016 (2016), Article ID 7843951, 9 pagesJournal of Drug Delivery Volume 2016 (2016), Article ID 7843951, 9 pages J. Mater. Chem., 2012, 22, 19964-19973.J. Mater. Chem., 2012, 22, 19964-19973.

본 발명의 목적은 케라틴을 유효성분으로 포함하는 간 보호용 약학적 조성물 및 건강기능식품 조성물을 제공하기 위한 것이다.It is an object of the present invention to provide a pharmaceutical composition for liver protection and a health functional food composition comprising keratin as an active ingredient.

또한, 본 발명의 다른 목적은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간 보호용 약학적 조성물 및 건강기능식품 조성물을 제공하기 위한 것이다.Another object of the present invention is to provide a pharmaceutical composition for liver protection comprising a polyethylene glycol (PEGylated) keratin nanoparticle as an active ingredient and a health functional food composition.

또한, 본 발명의 다른 목적은 케라틴을 유효성분으로 포함하는 간독성 질환 예방 또는 치료용 약학적 조성물, 및 간독성 질환 예방 또는 개선용 건강기능식품 조성물을 제공하기 위한 것이다. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatotoxic diseases comprising keratin as an active ingredient, and a health functional food composition for preventing or improving hepatotoxicity.

또한, 본 발명의 다른 목적은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간독성 질환 예방 또는 치료용 약학적 조성물, 및 간독성 질환 예방 또는 개선용 건강기능식품 조성물을 제공하기 위한 것이다. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatotoxic diseases comprising polyethylene glycol (PEGylated) keratin nanoparticles as an active ingredient, and a health functional food composition for preventing or improving hepatotoxicity .

상기 목적을 달성하기 위한 하나의 양태로서 본 발명은 케라틴을 유효성분으로 포함하는 간 보호용 약학적 조성물을 제공한다. In one aspect of the present invention, there is provided a pharmaceutical composition for liver protection comprising keratin as an active ingredient.

본 발명에 있어서, 상기 케라틴은 상업적으로 이용가능한 임의의 케라틴일 수 있으나, 바람직하게 머리카락 유래의 케라틴이고, 예를 들어 사람 머리카락으로부터 수득한 케라틴일 수 있다. In the present invention, the keratin may be any keratin which is commercially available, but is preferably keratin derived from hair, for example, keratin obtained from human hair.

구체적으로 본 발명의 케라틴은 사람 머리카락을 클로로포름과 메탄올을 2:1의 비율로 섞은 혼합액에 넣어 지질을 제거하고, 2 % 과산화아세트산 용액에서 12시간 교반한 후, 5 % 2-머캅토에탄올, 5 M 우레아, 2.6 M 티오우레아 및 25 mM Tris-HCl(pH 8.5)에 넣고, 50℃에서 72시간동안 반응시켜 수득할 수 있다.Specifically, the keratin of the present invention is prepared by adding human hair to a mixture of chloroform and methanol in a ratio of 2: 1 to remove lipid, stirring the mixture in a 2% peracetic acid solution for 12 hours, adding 5% 2-mercaptoethanol, 5 M urea, 2.6 M thiourea and 25 mM Tris-HCl (pH 8.5), and reacted at 50 캜 for 72 hours.

또한, 본 발명에 있어서, 상기 케라틴은 바람직하게 폴리에틸렌글리콜(polyethyleneglycol, PEG)화된(PEGylated) 케라틴일 수 있다. Further, in the present invention, the keratin may preferably be a polyethyleneglycol (PEGylated) keratin.

상기 PEG화된 케라틴이란 케라틴의 아미노산 잔기에 PEG가 결합되어 있는 물질을 의미한다. 본 발명의 PEG화된 케라틴에 있어서, PEG는 케라틴의 임의의 아미노산 잔기에 제한 없이 결합될 수 있으나, 바람직하게는 아민기에 결합한다.The PEGylated keratin means a substance in which PEG is bonded to an amino acid residue of keratin. In the PEGylated keratin of the present invention, the PEG may bind to any amino acid residue of the keratin unrestrictedly, but preferably binds to an amine group.

본 발명에서, PEG는 예를 들어, O-메틸-O`-숙시닐 폴리에틸렌글리콜, 2염기산 폴리에틸렌글리콜, α,ω-비스(2-카르복시에틸-폴리에틸렌글리콜, O,O`-비스(2-브로모에틸)폴리에틸렌글리콜, O,O`-비스(2-클로로에틸)폴리에틸렌글리콜, 폴리에틸렌 글리콜 디메실산, 메톡시폴리에틸렌글리콜 아세트산, O-[2-(3-숙시닐아미노)에틸]-O`-메틸-폴리에틸렌글리콜, O-(2-브로모에틸)-O`-메틸폴리에틸렌글리콜, O-(2-클로로에틸)-O`-메틸폴리에틸렌글리콜, 폴리에틸렌글리콜 모노메틸 에테르 메실산(Mesylate), 알데히드 기능화 폴리에틸렌글리콜, 글리시딜이써 기능화 폴리에틸렌글리콜, 나이트로페닐 카보네이트 기능화 폴리에틸렌글리콜, 메실(Mesyl) 기능화 폴리에틸렌글리콜 또는 토실(Tosyl) 기능화 폴리에틸렌글리콜일 수 있으며, 바람직하게는 O-메틸-O`-숙시닐 폴리에틸렌 글리콜일 수 있다. 상기 PEG의 분자량은 1000 내지 20000일 수 있으며, 바람직하게는 2000 내지 10000 일 수 있다.In the present invention, the PEG is, for example, O-methyl-O'-succinylpolyethyleneglycol, dibasic acid polyethylene glycol, alpha, omega- bis (2- carboxyethyl-polyethylene glycol, O, (2-chloroethyl) polyethylene glycol, polyethylene glycol dimesyl acid, methoxypolyethylene glycol acetic acid, O- [2- (3-succinylamino) ethyl] - O-methyl polyethyleneglycol, O- (2-bromoethyl) -O'-methylpolyethylene glycol, O- (2-chloroethyl) -O'- methylpolyethylene glycol, polyethylene glycol monomethyl ether mesylate ), Aldehyde-functionalized polyethylene glycols, glycidyl ether functionalized polyethylene glycols, nitrophenyl carbonate functionalized polyethylene glycols, mesyl-functionalized polyethylene glycols or Tosyl-functionalized polyethylene glycols, preferably O- O'-Succinylpolyethylene Glycine It may kolil. The molecular weight of the PEG may be a 1,000 to 20,000, and may be preferably from 2,000 to 10,000.

케라틴은 높은 생체 적합성을 가지고 있으나, 비교적 낮은 용해도에 의해 생체재료로의 사용에 제약이 있다. 이에 본 발명의 조성물에는 필요에 따라 케라틴에 비하여 용해도가 훨씬 높은 PEG화된 케라틴을 사용할 수 있다. Although keratin has high biocompatibility, its use as a biomaterial is limited by its relatively low solubility. Thus, the composition of the present invention can use PEGylated keratin, which has a solubility higher than that of keratin, if necessary.

본 발명의 PEG화된 케라틴은, 예를 들어, O-메틸-O`-숙시닐 폴리에틸렌글리콜 5000 용액에 4-(4,6-디메톡시-1,3,5-트리아진-2-일) 4-메톡시몰폴리늄 클로라이드(DMTMM)를 첨가하여 교반한 후, 케라틴을 첨가하여 제조할 수 있다.The PEGylated keratin of the present invention can be prepared by, for example, adding 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) 4 -Methoxymoluronium chloride (DMTMM), and then adding keratin.

본 발명의 케라틴 또는 PEG화된 케라틴은 간에서 UGT1A1을 활성화시키는 것을 특징으로 한다. 상기 UGT1A1은 다양한 약물, 독성물질 등을 글루쿠론산화 하여 체외로 배출시키는 역할을 하며, 빌리루빈을 대사시켜 체외로 배출시키는 유일한 효소로 알려져 있다. The keratin or PEGylated keratin of the present invention is characterized by activating UGT1A1 in the liver. The UGT1A1 is known to be the only enzyme that excretes various drugs, toxic substances and the like by glucuronidase excretion and metabolizes bilirubin and excretes it out of the body.

또한, 본 발명의 케라틴 또는 PEG화된 케라틴은 UGT1A1 효소 및 UGT1A1과 관련된 수용체인 CAR (Constitutive Androstane Receptor), GR (Glucocorticoid Receptor), AhR (Aryl Hydrocarbon Receptor) 등의 발현을 향상시키며, AST (aspartate amino transferase) 및 ALT (alanine amino transferase) 수치를 효과적으로 낮추고, 동시에 GSH (glutathione) 수치를 상승시킨다. In addition, the keratin or PEGylated keratin of the present invention enhances the expression of UGT1A1 enzyme and UGT1A1 related receptors such as CAR (Constitutive Androstane Receptor), GR (Glucocorticoid Receptor) and AhR (Aryl Hydrocarbon Receptor) ) And alanine amino transferase (ALT) levels, while simultaneously elevating GSH (glutathione) levels.

구체적인 실험예에서, 케라틴을 간세포(hepatocyte)에 처리하고, UGT1A1의 mRNA 발현, 분자 발현 및 다양한 시토졸 수용체의 발현을 실시간 qPCR 및 면역세포화학 염색으로 확인하였다. 그 결과, 대조군에 비하여 케라틴 처리군은 UGT1A1의 mRNA 및 분자 발현을 현저하게 상승시켰고, UGT1A1의 시토졸 수용제인 AhR 및 GR의 발현 역시 현저하게 상승시켰다(도 6 내지 8).In a specific experimental example, keratin was treated with hepatocyte and mRNA expression of UGT1A1, molecular expression and expression of various cytosolic receptors were confirmed by real-time qPCR and immunocytochemical staining. As a result, the keratin-treated group significantly increased mRNA and molecular expression of UGT1A1 and significantly increased the expression of AhR and GR as cytosol receptors of UGT1A1 (Figs. 6 to 8).

또한, 구체적인 실험예에서, PEG화된 케라틴을 간세포(hepatocyte)에 처리하고, UGT1A1의 mRNA 발현 및 다양한 시토졸 수용체의 발현을 실시간 qPCR로 확인하였다. 그 결과, 대조군에 비하여 PEG화된 케라틴 처리군은 UGT1A1의 mRNA 발현을 현저하게 상승시켰고, UGT1A1의 시토졸 수용제인 AhR, CAR 및 GR의 발현 역시 현저하게 상승시켰다(도 9 내지 10).Also, in a specific example, PEGylated keratin was treated in hepatocytes and mRNA expression of UGT1A1 and expression of various cytosolic receptors were confirmed by real-time qPCR. As a result, the PEGylated keratin-treated group significantly increased UGT1A1 mRNA expression and the expression of UGT1A1 cytosolic receptors AhR, CAR, and GR significantly (Figs. 9-10).

또한, 구체적일 실험예에서, PEG화된 케라틴을 알코올 유도성 간 손상 동물모델에 투여한 결과, 간 손상, 간 독성에 의해 높아진 AST (aspartate amino transferase) 및 ALT (alanine amino transferase) 효소들의 활성도가 PEG화된 케라틴 투여군에서는 낮아지는 것이 관찰되었다. 또한 에탄올로 유발된 간 손상, 독성에 의해 높아진 혈중 콜레스테롤의 농도가 PEG화된 케라틴 투여군에서 낮아졌고, 간 손상시 저하되는 글루타티온(GSH)의 농도가 PEG화된 케라틴 투여군에서 정상치로 회복되는 것을 관찰할 수 있었다(도 11). In addition, in a specific example, PEGylated keratin was administered to an animal model of alcohol-induced liver damage, and the activities of aspartate amino transferase (AST) and alanine amino transferase (ALT) And decreased in the keratin treated group. In addition, the concentration of blood cholesterol increased by ethanol - induced liver injury and toxicity was lowered in the PEGylated keratin - treated group, and the concentration of glutathione (GSH), which decreased in liver injury, recovered to the normal value in the PEGylated keratin - treated group (Fig. 11).

위와 같이, 본 발명의 케라틴 또는 PEG화된 케라틴은 글루쿠론산화에 과정에 연관된, UGT1A1 관련 상태 또는 질병에 대한 예방, 치료 효과를 가지며, 약물, 독성물질, 빌리루빈, 안드로젠, 에스트로젠, 코르티코이드, 지방산 유도체, 레티노이드 및 답즘산 등을 글루쿠론산화에 의해 배출 촉진시킬 수 있고, 이에 따라 빌리루빈의 축적이나, 간 손상, 간 독성을 해소할 수 있다. As described above, the keratin or PEGylated keratin of the present invention has a preventive and therapeutic effect on a UGT1A1-related condition or disease, which is related to the process of glucuron oxidation, and can be used as a drug, a toxic substance, a bilirubin, androgen, an estrogen, a corticoid, Retinoids and acyltransferases can be promoted by glucuronidation, and thus the accumulation of bilirubin, liver damage and liver toxicity can be solved.

또한, 본 발명의 케라틴 또는 PEG화된 케라틴은 간 손상의 지표로 알려진 ALT, AST 수치를 낮추고 GSH의 수치를 정상으로 회복시키는, 간세포 보호 효과 및 간독성 억제효과를 가진다. In addition, the keratin or PEGylated keratin of the present invention has hepatocyte protective effect and hepatotoxicity inhibiting effect, which lower ALT and AST levels, which are known as indicators of liver damage, and restore normal levels of GSH.

따라서, 본 발명의 케라틴 또는 PEG화된 케라틴을 유효성분으로 포함하는 약학적 조성물은 간 보호용 조성물 또는 간독성 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다. Accordingly, the pharmaceutical composition comprising the keratin or PEGylated keratin as an active ingredient of the present invention can be usefully used as a composition for protecting the liver or a pharmaceutical composition for the prevention or treatment of hepatotoxic diseases.

본 발명에 있어서, 상기 간독성 질환은 이에 제한되지는 않으나 예를 들어, 약물성 간 손상, 바이러스성 간 손상, 간염, 간경화, 간암 또는 간성혼수일 수 있다. In the present invention, the hepatotoxic diseases include, but are not limited to, drug-induced liver damage, viral liver damage, hepatitis, liver cirrhosis, liver cancer or hepatic coma.

다른 하나의 양태로서, 본 발명은 폴리에틸렌글리콜화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간 보호용 약학적 조성물을 제공한다. In another aspect, the present invention provides a pharmaceutical composition for liver protection comprising polyethylene glycolized (PEGylated) keratin nanoparticles as an active ingredient.

상기 폴리에틸렌글리콜화, 즉 PEG화된 케라틴 및 이의 제조방법은 앞서 설명한 바와 같다. The polyethylene glycolization, that is, the PEGylated keratin and the preparation method thereof are as described above.

본 발명의 PEG화된 케라틴은 나노입자 형태로 제조될 수 있는데, 나노입자 형성에 사용하는 용매로는, 증류수, 이차수, 삼차수, 인산완충식염수, 정제수, 멸균수, C1 내지 C4 알코올 또는 에틸렌 글라이콜 등을 들 수 있으나, 이에 제한되는 것은 아니며, 바람직하게는 삼차수 또는 인산완충식염수일 수 있다. The PEGylated keratin of the present invention can be prepared in the form of nanoparticles. Examples of the solvent used for forming nanoparticles include distilled water, secondary water, tertiary water, phosphate buffered saline, purified water, sterilized water, C 1 to C 4 alcohol or ethylene Glycol, and the like, but it is not limited thereto, and preferably it may be a tertiary water or a phosphate buffered saline.

본 발명의 나노입자 형성 시, PEG화된 케라틴은 0.2 (w/v)% 이상, 바람직하게는 0.2 (w/v)% 이상 5 (w/v)% 이하의 농도로 상기 용매에 첨가될 수 있으며, 보다 바람직하게는 0.2 (w/v)% 이상 0.5 (w/v)% 이하의 농도로 첨가될 수 있다.In forming nanoparticles of the present invention, the PEGylated keratin may be added to the solvent at a concentration of 0.2 (w / v)% or more, preferably 0.2 (w / v)% to 5 (w / v) , More preferably 0.2 (w / v)% to 0.5 (w / v)% or less.

본 발명의 PEG화된 케라틴 나노입자의 크기는 PEG화된 케라틴이 첨가된 용액의 볼텍스, 교반 또는 음파처리 속도에 따라 조절될 수 있으며, 바람직하게는 20 nm 내지 500 nm, 더 바람직하게는 60 nm 내지 100 nm 일 수 있다.The size of the PEGylated keratin nanoparticles of the present invention can be controlled by the vortex, stirring or sonication rate of the solution to which the PEGylated keratin is added, preferably from 20 nm to 500 nm, more preferably from 60 nm to 100 nm nm.

본 발명의 PEG화된 케라틴 나노입자는 예를 들어, PEG화된 케라틴을 2 mg/ml 이상의 농도로 pH 7.4의 PBS에 가하여 볼텍스, 교반 또는 음파처리함으로써, PEG화된 케라틴으로 구성된 나노입자를 형성시킬 수 있다. The PEGylated keratin nanoparticles of the present invention can be formed, for example, by adding a PEGylated keratin to a PBS at pH 7.4 at a concentration of 2 mg / ml or greater and vortexing, stirring or sonicating to form nanoparticles composed of PEGylated keratin .

또한, 본 발명의 상기 PEG화된 케라틴은 마이셀 나노입자를 형성하여 생리활성물질, 약물 등을 담지할 수 있는데, 용해도가 낮은 소수성 약물도 효과적으로 담지할 수 있다. 예를 들어, PEG화된 케라틴 나노입자는 본 발명의 PEG화된 케라틴을 중성 범위의 수계 용액, 구체적으로는 pH 6 내지 pH 9, 바람직하게는 pH 7 내지 pH 8, 가장 바람직하게는 pH 7 내지 pH 7.5의 수계 용매에 첨가하여 제조할 수 있다.In addition, the PEGylated keratin of the present invention can support physiologically active substances, drugs and the like by forming micellar nanoparticles, and can also carry hydrophobic drugs having low solubility effectively. For example, PEGylated keratin nanoparticles can be prepared by reacting the PEGylated keratin of the present invention with a neutral range of aqueous solutions, specifically pH 6 to pH 9, preferably pH 7 to pH 8, most preferably pH 7 to pH 7.5 To an aqueous solvent of water.

본 발명의 PEG화된 케라틴 나노입자에 담지되는 약물 또는 생리활성물질은, 이에 제한되는 것은 아니나, UGT1A1 관련 부작용을 가진 약물 또는 생리활성물질일 수 있다. The drug or physiologically active substance to be carried on the PEGylated keratin nanoparticle of the present invention may be a drug or a physiologically active substance having a side effect related to UGT1A1, but not limited thereto.

바람직하게 UGT1A1 관련 부작용을 가진 약물 또는 생리활성물질은 고빌리루빈 혈증, 빌리루빈 유도성 뇌기능 장애, 용혈성 황달 또는 간독성과 같은 간 손상 또는 간기능 장애를 유발하거나, 혹은 부작용으로 가지거나, 가질 수 있는 약물 또는 생리활성물질로서, 예를 들어, 아타자나비어(atazanavir), 세프트리악손(ceftriaxone), 아세트아미노펜(acetaminophen), 벨리노스타트(belinostat), 이리노테칸(irinotecan), 세팔로스포린(cephalosporin), 답손(dapsone), 레보도파(levodopa), 레보플록사신(levofloxacin), 메틸도파(methyldopa), 니트로푸란토인(nitrofurantoin), 페니실린(penicillin), 바클로펜(baclofen), 클로나제팜(clonazepam), 트리헥실페니딜(trihexylphenidyl), 부프레노르핀(buprenorphine), 카르베딜롤(carvedilol), 독소루비신(doxorubicin), 에티닐에스트라디올(ethinylestradiol), 엔타카폰(entacapone), SN-38, 에토포시드(etoposide), 에제티미브(ezetimibe), 몰핀(morphine), 날롤핀(nalorphine), 날트렉손(naltrexone), 레티가빈(retigabine), 트로포테칸(tropotecan) 및 트로글리타존(troglitazone)으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.Preferably, the drug or physiologically active substance having a UGT1A1-related adverse effect is a drug capable of causing or causing liver damage or liver dysfunction such as hyperbilirubinemia, bilirubin induced brain dysfunction, hemolytic jaundice or hepatotoxicity, Or physiologically active substances such as atazanavir, ceftriaxone, acetaminophen, belinostat, irinotecan, cephalosporin, but are not limited to, dapsone, levodopa, levofloxacin, methyldopa, nitrofurantoin, penicillin, baclofen, clonazepam, trihexylpyridyl, but are not limited to, trihexylphenidyl, buprenorphine, carvedilol, doxorubicin, ethinylestradiol, entacapone, SN-38, And is selected from the group consisting of etoposide, ezetimibe, morphine, nalorphine, naltrexone, retigabine, tropotecan and troglitazone. It can be any one or more.

상기 약물 또는 생리활성물질의 나노입자 내 담지량은 5 중량% 내지 30 중량% 일 수 있으며, 바람직하게는 10 중량% 내지 20 중량% 일 수 있다.The amount of the drug or physiologically active substance carried in the nanoparticles may be 5 wt% to 30 wt%, preferably 10 wt% to 20 wt%.

본 발명의 구체적인 일 실시예에서, PEG화된 케라틴을 pH 9.0의 삼차수에 용해시키고, 낮아진 pH를 pH 9.0로 다시 조절한 후, 염화칼슘 용액(pH 9.0)을 첨가한 후 교반하고, 이리노테칸 염산염 용액(pH 9.0)을 첨가한 후 교반하고, 탄산나트륨 용액(pH 9.0)을 첨가한 후 교반하여, 이리노테칸을 담지하는 PEG화된 케라틴 나노입자를 수득하였다.In a specific embodiment of the present invention, the PEGylated keratin is dissolved in a tertiary water of pH 9.0, the lowered pH is again adjusted to pH 9.0, then the calcium chloride solution (pH 9.0) is added and stirred, and the irinotecan hydrochloride solution pH 9.0) was added thereto, and the mixture was stirred. Then, sodium carbonate solution (pH 9.0) was added and stirred to obtain PEGylated keratin nanoparticles carrying irinotecan.

본 발명의 케라틴은 생체적합성이 우수하고, 독성이 없으며, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자 역시 세포독성을 거의 나타내지 않는다. The keratin of the present invention is excellent in biocompatibility, is not toxic, and PEGylated keratin or PEGylated keratin nanoparticles show little cytotoxicity.

본 발명의 조성물은 본 발명의 효과를 해치지 않는 범위 안에서 약학적으로 허용 가능한 희석제, 결합제, 붕해제, 윤활제, pH 조절제, 산화방지제, 용해보조제 등의 첨가제를 포함할 수 있다.The composition of the present invention may contain additives such as pharmaceutically acceptable diluents, binders, disintegrants, lubricants, pH adjusting agents, antioxidants, solubilizing aids and the like within the range not impairing the effects of the present invention.

희석제는 슈가, 전분, 미결정셀룰로오스, 유당(유당수화물), 포도당, 디-만니톨, 알기네이트, 알칼리토금속류염, 클레이, 폴리에틸렌글리콜, 무수인산수소칼슘, 또는 이들의 혼합물 등을 사용할 수 있다.The diluent may be sugar, starch, microcrystalline cellulose, lactose (lactose hydrate), glucose, di-mannitol, alginate, alkaline earth metal salt, clay, polyethylene glycol, anhydrous calcium hydrogen phosphate or a mixture thereof.

결합제는 전분, 미결정셀룰로오스, 고분산성 실리카, 만니톨, 디-만니톨, 자당, 유당수화물, 폴리에틸렌글리콜, 폴리비닐피롤리돈(포비돈), 폴리비닐피롤리돈 공중합체(코포비돈), 히프로멜로오스, 히드록시프로필셀룰로오스, 천연검, 합성검, 젤라틴, 또는 이들의 혼합물 등을 사용할 수 있다.The binder may be selected from the group consisting of starch, microcrystalline cellulose, highly disperse silica, mannitol, di-mannitol, sucrose, lactose hydrate, polyethylene glycol, polyvinylpyrrolidone (povidone), polyvinylpyrrolidone copolymer , Hydroxypropylcellulose, natural gum, synthetic gum, gelatin, or a mixture thereof.

붕해제는 전분글리콘산나트륨, 옥수수전분, 감자전분 또는 전호화전분 등의 전분 또는 변성전분; 벤토나이트, 몬모릴로나이트, 또는 비검(veegum) 등의 클레이; 미결정셀룰로오스, 히드록시프로필셀룰로오스 또는 카르복시메틸셀룰로오스 등의 셀룰로오스류; 알긴산나트륨 또는 알긴산 등의 알긴류; 크로스카멜로스(croscarmellose) 나트륨 등의 가교 셀룰로오스류; 구아검, 잔탄검 등의 검류; 가교 폴리비닐피롤리돈(crospovidone) 등의 가교 중합체; 중탄산나트륨, 시트르산 등의 비등성 제제, 또는 이들의 혼합물을 사용할 수 있다.The disintegrant may be a starch or modified starch such as sodium starch glycolate, corn starch, potato starch or pregelatinized starch; Clays such as bentonite, montmorillonite, or veegum; Cellulose such as microcrystalline cellulose, hydroxypropylcellulose or carboxymethylcellulose; Alginates such as sodium alginate and alginic acid; Crosslinked celluloses such as croscarmellose sodium; Guar gum and xanthan gum; Crosslinked polymers such as crosslinked polyvinylpyrrolidone (crospovidone); Sodium bicarbonate, citric acid and the like, or a mixture thereof.

윤활제는 탈크, 스테아린산, 스테아린산 마그네슘, 스테아린산 칼슘, 라우릴 설페이트나트륨, 수소화식물성오일, 나트륨벤조에이트, 나트륨스테아릴푸마레이트, 글리세릴 베헤네이트, 글리세릴 모노레이트, 글리세릴모노스테아레이트, 글리세릴 팔미토스테아레이트, 콜로이드성 이산화규소 또는 이들의 혼합물 등을 사용할 수 있다.The lubricant may be selected from the group consisting of talc, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, sodium stearyl fumarate, glyceryl behenate, glyceryl monolate, glyceryl monostearate, Stearate, colloidal silicon dioxide, or a mixture thereof may be used.

pH 조절제는 초산, 아디프산, 아스코르빈산, 아스코르빈산 나트륨, 에테르산 나트륨, 사과산, 숙신산, 주석산, 푸마르산, 구연산(시트르산)과 같은 산성화제와 침강 탄산 칼슘, 암모니아수, 메글루민, 탄산 나트륨, 산화 마그네슘, 탄산 마그네슘, 구연산 나트륨, 삼염기칼슘인산염과 같은 염기성화제 등을 사용할 수 있다.The pH adjusting agent may be an acidifying agent such as acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium ethoxide, malic acid, succinic acid, tartaric acid, fumaric acid, citric acid (citric acid) and precipitated calcium carbonate, ammonia water, meglumine, Basic agents such as sodium, magnesium oxide, magnesium carbonate, sodium citrate, and tribasic calcium phosphate can be used.

산화방지제는 디부틸 히드록시 톨루엔, 부틸레이티드 히드록시아니솔, 초산토코페롤, 토코페롤, 프로필 갈레이트, 아황산수소나트륨, 피로아황산나트륨 등을 사용할 수 있다.As the antioxidant, dibutylhydroxytoluene, butylated hydroxyanisole, tocopheryl acetate, tocopherol, propyl gallate, sodium hydrogen sulfite, sodium pyroa sulfate and the like can be used.

용해보조제는 라우릴황산나트륨, 폴리소르베이트 등의 폴리옥시에틸렌 소르비탄 지방산 에스테류, 도큐세이트 나트륨, 폴록사머(poloxamer) 등을 사용할 수 있다.Solubilizing adjuvants include polyoxyethylene sorbitan fatty acid esters such as sodium lauryl sulfate and polysorbate, docusate sodium, poloxamer, and the like.

본 발명의 조성물은 경구투여를 위하여 정제, 환제, 산제, 과립제, 캡슐제 등의 고형제제로 제제화될 수 있으며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 제조될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 함께 사용될 수 있다. 또한 상기 약학적 조성물이 경구를 위한 현탁제, 내용액제, 유제, 시럽제 등의 액상제제로 제제화될 수 있으며, 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 사용하여 액상제제로 제제화될 수 있다. The composition of the present invention may be formulated as a solid preparation for oral administration, such as tablets, pills, powders, granules, capsules, etc. Such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. The pharmaceutical composition may be formulated into liquid preparations such as suspensions, solutions, emulsions and syrups for oral use. In addition to water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be used And can be formulated into liquid formulations.

또한, 본 발명의 조성물은 비경구 투여를 위한 제제화를 위하여 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. In addition, the composition of the present invention may contain a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, and a suppository for formulation for parenteral administration. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

상기 조성물의 투여량은, 담지된 약물의 종류, 환자의 중증, 나이, 성별, 체중 등의 환자의 상태와 투여경로 및 투여 기간에 따라 적절하게 조절될 수 있다. The dosage of the composition can be appropriately adjusted depending on the condition of the patient such as the type of the drug, the severity of the patient, age, sex, weight, etc., and the administration route and administration period.

상기 용어 투여는 어떠한 적절한 방법으로 환자에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 본 발명의 약학적 조성물의 투여 경로는 목적조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 경구투여, 복강 내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 비내 투여, 폐내 투여, 직장내 투여, 강내 투여, 복강내 투여, 경막내 투여될 수 있으나, 이에 한정되지는 않는다. 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.Administration of the term means introducing the pharmaceutical composition of the present invention to the patient in any suitable manner and the administration route of the pharmaceutical composition of the present invention can be administered through any conventional route so long as it can reach the target tissue . But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrathecal administration, intraperitoneal administration, intraperitoneal administration and intraperitoneal administration . For example, by oral, rectal or intravenous, intramuscular, subcutaneous, intramural or intracerebroventricular injection.

본 발명의 약학적 조성물에서, 케라틴, PEG화된 케라틴, 또는 PEG화된 케라틴 나노입자는 총 조성물의 중량에 대하여 0.001 %(w/v) 내지 10 %(w/v), 바람직하게는 0.01 %(w/v) 내지 5 %(w/v), 더욱 바람직하게는 0.05 %(w/v) 내지 2 %(w/v)의 함량으로 포함될 수 있다. In a pharmaceutical composition of the present invention, the keratin, PEGylated keratin, or PEGylated keratin nanoparticles are present in an amount of from 0.001% (w / v) to 10% (w / v), preferably 0.01% / v) to 5% (w / v), more preferably 0.05% (w / v) to 2% (w / v).

본 발명에 따른 조성물은 추가적인 활성성분을 더 포함할 수 있으며, 단독 또는 호르몬 치료, 약물 치료 등의 다양한 방법들과 병용하여 사용될 수 있다.The composition according to the present invention may further comprise additional active ingredients and may be used alone or in combination with various methods such as hormone therapy, drug therapy, and the like.

또한, 다른 양태로서, 본 발명은 케라틴을 유효성분으로 포함하는 간 보호용 건강기능식품 조성물, PEG화된 케라틴을 유효성분으로 포함하는 간 보호용 건강기능식품 조성물, 및 PEG화된 케라틴 나노입자를 유효성분으로 포함하는 간 보호용 건강기능식품 조성물을 제공한다.In addition, in another aspect, the present invention provides a health functional food composition for liver protection comprising keratin as an active ingredient, a health functional food composition for liver protection comprising PEGylated keratin as an active ingredient, and a pegylated keratin nanoparticle as an active ingredient And a health functional food composition for protecting the liver.

또한, 다른 양태로서, 본 발명은 케라틴을 유효성분으로 포함하는 간독성 질환 예방 또는 개선용 건강기능식품 조성물, PEG화된 케라틴을 유효성분으로 포함하는 간독성 질환 예방 또는 개선용 건강기능식품 조성물, 및 PEG화된 케라틴 나노입자를 유효성분으로 포함하는 간독성 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, in another aspect, the present invention provides a health functional food composition for preventing or ameliorating hepatotoxic diseases comprising keratin as an active ingredient, a health functional food composition for preventing or improving hepatotoxic diseases comprising PEGylated keratin as an active ingredient, A health functional food composition comprising keratin nanoparticles as an active ingredient for preventing or improving hepatotoxic diseases.

상기 케라틴, PEG화된 케라틴, PEG화된 케라틴 나노입자, 간 보호, 간독성 질환은 앞서 설명한 바와 같다. The keratin, PEGylated keratin, PEGylated keratin nanoparticles, liver protection, hepatotoxic diseases are as described above.

본 발명의 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자를 유효성분으로 포함하는 건강기능식품 조성물은 간 보호용, 또는 간독성 질환의 예방 및 개선을 위한 약제, 식품 및 음료 등에 다양하게 이용될 수 있다.The health functional food composition comprising the keratin, PEGylated keratin or PEGylated keratin nanoparticles of the present invention as an active ingredient can be used for various purposes in medicines, foods and beverages for the protection of liver or for the prevention and improvement of hepatotoxic diseases.

본 발명의 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자를 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 침출차, 건강보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.Examples of foods to which the keratin, PEGylated keratin or PEGylated keratin nanoparticles of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, leached tea, health supplement foods, Granules, tablets, capsules or beverages.

또한, 본 발명의 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자는 간 보호 및 간 질환의 예방 및 개선 효과를 갖는 식품 또는 식품첨가제로 제공될 수 있다. In addition, the keratin, PEGylated keratin or PEGylated keratin nanoparticles of the present invention can be provided as a food or food additive having an effect of preventing and improving liver protection and liver disease.

본 발명의 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자를 첨가할 수 있는 식품 형태는 캔디류의 각종 식품류, 음료, 껌, 차, 비타민 복합제, 또는 건강보조 식품류인 식품 등을 포함한다.The food forms to which the keratin, PEGylated keratin or PEGylated keratin nanoparticles of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.

상기 외에 본 발명의 조성물은 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, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

본 발명의 케라틴, PEG화된 케라틴 또는 PEG화된 케라틴 나노입자는 독성이 적거나 거의 없고, UGT1A1 및 관련 수용체의 발현을 현저하게 상승시키며, 간 손상의 지표인 ALT 및 AST의 수치를 낮추고, GSH 수치를 증가시킴으로써, 간세포 보호작용, 간독성 억제작용을 가지므로, 간 보호용 조성물 또는 간독성 질환의 예방 또는 치료용 조성물로 유용하게 사용될 수 있다. The keratin, PEGylated keratin or PEGylated keratin nanoparticles of the present invention have little or no toxicity, significantly increase the expression of UGT1A1 and related receptors, lower levels of ALT and AST, indicators of liver damage, and increase GSH levels , It has hepatocyte protective action and hepatotoxicity inhibiting action and thus can be effectively used as a composition for protecting the liver or a composition for preventing or treating hepatotoxic diseases.

도 1은 PEG화된 케라틴을 제조하는 방법을 도식화한 것이다.
도 2는 PEG화된 케라틴을 NMR 분석한 데이터이다.
도 3의 a는 PEG화된 케라틴 나노입자를 도식화한 것이고, b는 PEG화된 케라틴 나노입자의 평균 입자 크기를 동적 광산란으로 측정한 그래프이며, c는 PEG화된 케라틴 나노입자를 투과전자현미경(TEM)으로 관찰한 것이다.
도 4는 PEG화된 케라틴 나노입자의 평균 입자 크기를 동적 광산란으로 측정한 그래프와 PEG화된 케라틴 나노입자를 투과전자현미경(TEM)으로 관찰한 것을 나타낸 것이다.
도 5의 a는 PEG화된 케라틴 나노입자의 평균 입자 크기를 동적 광산란으로 측정한 그래프이며, b는 PEG화된 케라틴 나노입자를 투과전자현미경(TEM)으로 관찰한 것이며, c는 EDX (Energy-dispersive X-ray spectroscopy) 분석으로 PEG화된 케라틴 나노입자에 담지된 탄산칼슘을 확인한 것이고, d는 FT-IR(Fourier Transform Infrared) 분석으로 탄산칼슘의 바테라이트 결정형을 확인한 것이다.
도 6은 케라틴의 UGT1A1의 mRNA 발현을 실시간 qPCR로 확인한 결과이다.
도 7은 케라틴의 UGT1A1의 분자 발현을 면역세포화학 염색으로 확인한 결과이다.
도 8은 케라틴의 UGT1A1의 mRNA 발현을 실시간 qPCR로 확인한 결과이다.
도 9는 PEG화된 케라틴의 UGT1A1의 mRNA 발현을 실시간 qPCR로 확인한 결과이다.
도 10은 PEG화된 케라틴의 UGT1A1 관련 시토졸 수용체 발현을 실시간 qPCR로 확인한 결과이다.
도 11은 PEG화된 케라틴을 투여한 알코올 유도성 간 손상 동물모델을 희생시킨 후, 간 중량의 측정 결과, ALT, AST, 총 콜레스테롤 수치 및 간 조직의 총 GSH 수치를 확인한 결과를 나타낸 것이다. Figure 1 illustrates a method of making PEGylated keratin.
Fig. 2 is NMR data of PEGylated keratin.
FIG. 3 (a) is a graphical representation of the PEGylated keratin nanoparticles, (b) is the average particle size of the PEGylated keratin nanoparticles as measured by dynamic light scattering, and c is the PEGylated keratin nanoparticles as a transmission electron microscope .
FIG. 4 is a graph showing the average particle size of pegylated keratin nanoparticles measured by dynamic light scattering and a PEG keratin nanoparticle observed by transmission electron microscopy (TEM).
FIG. 5A is a graph showing the average particle size of pegylated keratin nanoparticles measured by dynamic light scattering, b is the observation of PEGylated keratin nanoparticles with a transmission electron microscope (TEM), c is energy-dispersive X -ray spectroscopy analysis of calcium carbonate supported on pegylated keratin nanoparticles, and d is the determination of calcium carbonate vaterite by FT-IR (Fourier Transform Infrared) analysis.
Fig. 6 shows the results of confirming mRNA expression of UGT1A1 of keratin by real-time qPCR.
Fig. 7 shows the result of confirming the molecular expression of UGT1A1 of keratin by immunocytochemistry.
Fig. 8 shows the result of confirming mRNA expression of UGT1A1 of keratin by real-time qPCR.
FIG. 9 shows the results of confirming the mRNA expression of PEGylated keratin UGT1A1 by real-time qPCR.
FIG. 10 shows the results of real-time qPCR analysis of UGT1A1-related cytosolic receptor expression of PEGylated keratin.
FIG. 11 shows results of liver weight measurement, ALT, AST, total cholesterol, and total GSH levels of liver tissues after sacrificing an alcohol-induced liver injury animal model to which PEGylated keratin was administered.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나, 이들 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명의 범위가 하기 내용에 의해 한정되는 것은 아니다. Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, these embodiments are provided only for the purpose of easier understanding of the present invention, and the scope of the present invention is not limited by the following description.

실시예Example 1: 케라틴의 제조 1: Manufacture of keratin

사람 머리카락을 약한 세제를 이용해서 깨끗하게 세척한 후 증류수로 여러 번 씻어주었다. 지질제거를 위해 머리카락을 비커에 담고, 클로로포름과 메탄올을 2:1의 비율로 섞은 혼합액에 머리카락이 잠길 때까지 넣어주었다. 24시간 후 지질 제거에 사용한 용액이 머리카락에 남아있지 않을 때까지 증류수로 여러 번 세척한 후 머리카락을 기건(air-dry)시켰다. 2 % 과산화아세트산(Sigma aldrich) 800 mL에 기건시킨 머리카락 20 g을 넣고 37℃에서 12시간동안 300 rpm의 속도로 교반하였다. 12시간 후, 머리카락을 체에 거르고 증류수로 세척하여 남아있는 산화물을 제거하였다. 머리카락을 400 mL 신다이(Shindai) 용액 (5 % 2-머캅토에탄올, 5 M 우레아, 2.6 M 티오우레아(이상 Sigma aldrich) 및 25 mM Tris-HCl(pH 8.5))에 넣고, 50 ℃, 400 rpm의 조건에서 72시간동안 반응시켰다. 그 후, 머리카락 용액을 50 mL 튜브에 넣고 3500 rpm으로 20분 동안 원심분리하였다. 상등액을 모아 12-14 kDa 컷오프 Spectra/Porⓡ4 투석막(Spectrum)을 이용해서 투석하였다. 투석이 끝난 케라틴 액체샘플을 동결 건조하여 케라틴 파우더를 제조하였다.Human hair was cleaned with weak detergent and washed several times with distilled water. For lipid removal, the hair was placed in a beaker and mixed with a mixture of chloroform and methanol at a ratio of 2: 1 until the hair was locked. After 24 hours, the solution used for lipid removal was washed several times with distilled water until the hair was not left in the hair, and the hair was air-dried. Twenty grams of hair fixed in 800 mL of 2% acetic acid (Sigma aldrich) was added and stirred at 37 rpm for 12 hours at 300 rpm. After 12 hours, the hair was sieved and washed with distilled water to remove remaining oxides. The hair was placed in a 400 mL Shindai solution (5% 2-mercaptoethanol, 5 M urea, 2.6 M thiourea and 25 mM Tris-HCl pH 8.5) For 72 hours. The hair solution was then placed in a 50 mL tube and centrifuged at 3500 rpm for 20 minutes. The supernatant was collected and dialyzed using a 12-14 kDa cut-off Spectra / Por ⓡ 4 dialysis membrane (Spectrum). The dialyzed keratin liquid sample was lyophilized to prepare a keratin powder.

실시예Example 2:  2: PEG화된PEGylated 케라틴의 제조 Manufacture of keratin

실시예Example 2-1:  2-1: PEG화된PEGylated 케라틴의 합성 Synthesis of keratin

도 1에 제시된 반응과정으로 PEG화된 케라틴을 합성하였다.PEGylated keratin was synthesized by the reaction process shown in FIG.

실시예 1에서 제조한 케라틴 파우더 0.5 g을 미세부품 단위계수 측정 전자저울(0.001 g ~ 620 g, AJ-620E)로 달아 500 mL 파이렉스 비이커(ISOLAB, YLS, 독일)에 넣었다. 비이커에 삼차수 100 mL를 넣고, 40×8 mm 마그네틱 교반봉([B00C3ME4ZA], 1572500 IKAFLON 40, IKA)과 MS-H-S10 10-Channel 마그네틱 교반기를 이용하여 300 rpm의 속도로 24시간 동안 교반하였다. 메톡시폴리에틸렌글리콜을 변형시킨 O-메틸-O`-숙시닐 폴리에틸렌글리콜 5000(mPEG, 17929-5G-F, Lot#R063737/2V Sigma aldrich) 300 mg을 삼차수 20 mL에 1시간 동안 녹였다. mPEG용액에 4-(4,6-디메톡시-1,3,5-트리아진-2-일) 4-메톡시몰폴리늄 클로라이드(DMTMM) n-수화물(327-53752, wako chemical) 16 mg을 넣고 1시간동안 교반하였다. 케라틴 용액과 mPEG 및 DMTMM의 결합 용액을 500 mL 비커에 넣어, 40×8 mm 마그네틱 교반봉(1572500 IKAFLON 40, IKA)과 MS-H-S10 10-Channel 마그네틱 교반기를 이용하여 500rpm의 속도로 실온에서 3일 동안 교반하여 PEG와 케라틴을 반응시킴으로써 PEG화된 케라틴을 제조하였다.0.5 g of the keratin powder prepared in Example 1 was weighed using an electronic balance (0.001 g to 620 g, AJ-620E) with a fine component unit count and placed in a 500 mL Pyrex beaker (ISOLAB, YLS, Germany). Add 100 mL of tertiary water to a beaker and stir at 300 rpm for 24 hours using a 40 × 8 mm magnetic stir bar (B00C3ME4ZA, 1572500 IKAFLON 40, IKA) and an MS-H-S10 10-Channel magnetic stirrer Respectively. 300 mg of O-methyl-O'-succinylpolyethylene glycol 5000 (mPEG, 17929-5G-F, Lot # R063737 / 2V Sigma aldrich) modified with methoxypolyethylene glycol was dissolved in 20 mL of tertiary water for 1 hour. To a solution of mPEG was added 16 mg of 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) 4- methoxymoluronium chloride (DMTMM) n-hydrate (327-53752, wako chemical) And the mixture was stirred for 1 hour. The keratin solution and the conjugate solution of mPEG and DMTMM were placed in a 500 mL beaker and heated at a rate of 500 rpm at room temperature using a 40 × 8 mm magnetic stir bar (1572500 IKAFLON 40, IKA) and an MS-H-S10 10-Channel magnetic stirrer PEGylated keratin was prepared by reacting PEG and keratin with stirring for 3 days.

실시예Example 2-2:  2-2: PEG화된PEGylated 케라틴의 정제 Purification of keratin

제조된 PEG화 케라틴을 Spectra/Por 투과막 튜브(직경 16 mm, 평폭 25 mm, MW10000, Spectrum)에 한 튜브당 100 mL씩 넣고, Spectra/Por RC 투석 튜빙 클로져(최대 폭 55 mm, Orange, Spectrum)를 양쪽 튜브에 패킹하였다. 5L 플라스틱 비이커(지름 197 mm, 높이 265 mm)에 삼차수 4 L를 채워 PEG화된 케라틴 용액이 담긴 튜브를 넣고 마그네틱 바를 넣고 교반하면서 투석하였다. 이때, 12시간마다 새로운 삼차수로 갈아주었으며, 실온에서 3일 동안 삼차수로 총 6번을 갈아주면서 투석하였다. 최종 PEG화된 케라틴 용액을 50 mL 원심관(17x120 mm, BD Falcon)에 40 mL 씩 나눠 담은 후, 초저온냉장고(Nihon freezer, Upright type, Single cooling type, 542ℓ)에서 -70 ℃로 24시간 동안 완전히 동결시켰다. 원심관 뚜껑을 열고, 동결건조기(ALPHA 2-4 LSC PLUS, Laboratory freeze dryers, Christ)에 넣고 85 ℃의 진공상태에서 후 완전히 파우더 형태가 되도록 3일 동안 건조시켰다.Porous keratin was added to Spectra / Por permeable membrane tube (diameter 16 mm, 25 mm square, MW 10000, Spectrum) in a volume of 100 mL per tube, and a Spectra / Por RC dialysis tubing closure ) Were packed into both tubes. A 5 L plastic beaker (197 mm in diameter, 265 mm in height) was filled with 4 L of cubic millimeter and placed in a tube containing the pegylated keratin solution and dialyzed under stirring with a magnetic bar. At this time, a new three-dimensional number was changed every 12 hours and dialyzed while changing a total of 6 times at room temperature for three days in a cubic number. The final pegylated keratin solution was placed in a 50 mL centrifuge tube (17x120 mm, BD Falcon) in an amount of 40 mL, and then completely frozen at -70 ° C in a cryocooler (Nihon freezer, Upright type, . The lid of the centrifuge tube was opened and placed in a freeze drier (ALPHA 2-4 LSC PLUS, Laboratory freeze dryers, Christ), dried at 85 ° C under vacuum, and then completely dried for 3 days in powder form.

실시예Example 2-3:  2-3: PEG화된PEGylated 케라틴의 확인 Confirmation of keratin

실시예 2-2에서 얻은 PEG화된 케라틴을 NMR 분석하였다. 그 결과, 도 2에 나타난 바와 같이 PEG화된 케라틴에서 각각 케라틴 및 PEG에서 특이적으로 나타나는 피크가 동시에 존재하고 있음을 확인하였다. The PEGylated keratin obtained in Example 2-2 was analyzed by NMR. As a result, as shown in Fig. 2, it was confirmed that peaks which are specifically present in keratin and PEG are simultaneously present in PEGylated keratin.

실시예Example 3:  3: PEG화된PEGylated 케라틴 나노입자의 제조-1 Preparation of keratin nanoparticles -1

실시예 2에서 제조한 PEG화된 케라틴을 2 mg/ml 농도로 pH 7.4의 인산완충식염수(PBS)에 첨가하여 교반하여 PEG화된 케라틴으로 구성된 나노입자를 형성시켰다.PEGylated keratin prepared in Example 2 was added to phosphate buffered saline (PBS) of pH 7.4 at a concentration of 2 mg / ml and stirred to form nanoparticles composed of PEGylated keratin.

위 방법으로 얻은 PEG화된 케라틴 나노입자의 평균 입자 크기(hydrodynamic diameter)를 동적 광산란을 통해 계산하였다. 그 결과, 도 3에 나타난 바와 같이, PEG화된 케라틴 나노입자의 평균 입자 크기가 약 65 내지 85 nm인 것을 확인하였다.The average particle size (hydrodynamic diameter) of the PEGylated keratin nanoparticles obtained by the above method was calculated by dynamic light scattering. As a result, as shown in Fig. 3, it was confirmed that the average particle size of the PEGylated keratin nanoparticles was about 65 to 85 nm.

실시예Example 4:  4: PEG화된PEGylated 케라틴 나노입자의 제조-2 Production of keratin nanoparticles-2

실시예 2에서 제조한 PEG화된 케라틴을 2 mg/ml의 농도로 pH 9.0의 인산완충식염수(PBS)에 첨가한후 20 rpm의 속도로 교반하여 녹인 후에, PEG화된 케라틴 용액에 1N HCl을 천천히 첨가하여 pH를 7.4까지 점차 감소시켜 PEG화된 케라틴으로 구성된 나노입자를 형성시켰다.PEGylated keratin prepared in Example 2 was added to phosphate buffered saline (PBS) of pH 9.0 at a concentration of 2 mg / ml, and the mixture was stirred at a speed of 20 rpm to dissolve the pegylated keratin. Then, 1 N HCl was slowly added to the pegylated keratin solution To gradually reduce the pH to 7.4 to form nanoparticles composed of pegylated keratin.

위 방법으로 얻은 PEG화된 케라틴 나노입자의 평균 입자 크기(hydrodynamic diameter)를 동적 광산란을 통해 계산하였다. 그 결과, 도 4에 나타난 바와 같이, PEG화된 케라틴나노입자의 평균 입자 크기는 약 85 nm인 것으로 확인되었다.The average particle size (hydrodynamic diameter) of the PEGylated keratin nanoparticles obtained by the above method was calculated by dynamic light scattering. As a result, as shown in Fig. 4, it was confirmed that the average particle size of the PEGylated keratin nanoparticles was about 85 nm.

실시예Example 5: 약물( 5: Drugs ( 이리노테칸Irinotecan )이 )this 담지된Supported PEG화된PEGylated 케라틴 나노입자의 제조 Preparation of keratin nanoparticles

앞선 실시예에서 제조한 PEG화된 케라틴(50 mg)을 pH 9.0의 삼차수 3 mL에 용해시켰다. PEG화된 케라틴이 용해되면서 pH가 6.0으로 낮아져 0.1 N NaOH를 넣어 다시 pH 9.0으로 높였다. 그리고 별도로 염화칼슘(CaCl2)(20 mg, 0.18 mmol)을 삼차수(pH 9.0) 1 mL에 완전히 용해시켰다. 두 수용액을 혼합시켜 2시간 동안 1200 rpm의 속도로 교반한 후 이리노테칸 염산염(20 mg, 0.032 mmol, Sigma Aldrich Co.)의 3차수(pH 9.0, 2 mL) 수용액을 첨가하고 2시간 더 1200 rpm의 속도로 교반하였다. 탄산나트륨(20 mg, 0.19 mmol)을 삼차수(pH 9.0) 1 mL에 용해시킨 후 상기 PEG화된 케라틴, 염화칼슘, 이리노테칸 염산염(Sigma Co.)이 혼합된 수용액에 첨가하여 12 시간 동안 1200 rpm의 속도로 실온에서 교반하였다. 반응 후 삼차수(pH 9.0) 33 mL 첨가하여 용액 양을 40 mL로 맞추고 1000 rpm으로 10분 동안 원심분리기를 작동시킨 후 하층에 가라앉은 침전물을 제거하고 상층액을 삼투막 멤브레인(분자량 제한: 25,000 g/mol)을 이용하여 4시간 동안 삼투교환을 하였다. 최종적으로 72시간 동안 동결건조하여 갈색의 파우더 형태로 이리노테칸 및 탄산칼슘을 담지하는 PEG화된 케라틴 나노입자를 얻었다(수율: 52%). PEGylated keratin (50 mg) prepared in the previous example was dissolved in 3 mL of tertiary aqueous solution at pH 9.0. PEGylated keratin dissolved and the pH was lowered to 6.0, and the pH was further raised to 9.0 with 0.1 N NaOH. Separately, calcium chloride (CaCl 2 ) (20 mg, 0.18 mmol) was completely dissolved in 1 mL of tertiary aqueous solution (pH 9.0). The two aqueous solutions were mixed and stirred for 2 hours at a speed of 1200 rpm. An aqueous solution of irinotecan hydrochloride (20 mg, 0.032 mmol, Sigma Aldrich Co.) in tertiary order (pH 9.0, 2 mL) Lt; / RTI > Sodium carbonate (20 mg, 0.19 mmol) was dissolved in 1 mL of tertiary aqueous solution (pH 9.0) and then added to an aqueous solution containing the PEGylated keratin, calcium chloride, irinotecan hydrochloride (Sigma Co.) mixed therein at 1200 rpm for 12 hours Stir at room temperature. After the reaction, 33 mL of tertiary water (pH 9.0) was added to adjust the amount of the solution to 40 mL, and the centrifugal separator was operated at 1000 rpm for 10 minutes. Then, the precipitate deposited on the lower layer was removed and the supernatant was passed through an osmotic membrane g / mol) for 4 hours. Finally, the mixture was lyophilized for 72 hours to obtain PEG-modified keratin nanoparticles carrying irinotecan and calcium carbonate in the form of brown powder (yield: 52%).

상기 나노입자 내 이리노테칸 담지량은 0.1 N HCl 수용액을 이용해 이리노테칸 담지 탄산칼슘 복합 나노입자를 완전히 용해시킨 후 UV-Vis 스펙트로포토미터를 이용하여 정량하였다. 이리노테칸 염산염의 농도-흡광도의 표준 검량선(calibration standard cruve)을 이용해 계산하였으며 15 중량%인 것을 확인하였다.The amount of irinotecan in the nanoparticles was determined by completely dissolving irinotecan-supported calcium carbonate composite nano-particles using 0.1 N HCl aqueous solution and then using a UV-Vis spectrophotometer. The calibration standard curve for the concentration-absorbance of irinotecan hydrochloride was calculated and found to be 15% by weight.

실험예Experimental Example 1: 나노입자의 물리적 특성 확인 1: Identification of physical properties of nanoparticles

실시예 5에서 얻은 약물(이리노테칸)을 담지한 PEG화된 케라틴 나노입자의 평균 입자 크기(hydrodynamic diameter)를 동적 광산란을 통해 계산하였다. 그 결과, 도 5a에 나타난 바와 같이, 상기 약물담지 PEG화된 케라틴 나노입자의 평균 입자 크기는 260.0 nm인 것으로 확인되었다. The average particle size (hydrodynamic diameter) of the PEGylated keratin nanoparticles carrying the drug (irinotecan) obtained in Example 5 was calculated by dynamic light scattering. As a result, as shown in FIG. 5A, it was confirmed that the average particle size of the drug-supported PEGylated keratin nanoparticles was 260.0 nm.

또한, 실시예 5에서 얻은 PEG화된 케라틴 나노입자를 투과 전자 현미경(TEM)으로 조사한 결과, 도 5b에 나타난 바와 같이, 구형의 균일한 나노입자가 형성되었음을 확인할 수 있었다. Further, the PEGylated keratin nanoparticles obtained in Example 5 were examined by a transmission electron microscope (TEM), and it was confirmed that spherical uniform nanoparticles were formed as shown in FIG. 5B.

또한, 도 5c 및 5d에 각각 나타난 바와 같이, PEG화된 케라틴 나노입자의 EDX(Energy-dispersive X-ray spectroscopy) 분석으로 탄산칼슘 원소 성분을 확인할 수 있었고, FT-IR(Fourier Transform Infrared) 분석결과 탄산칼슘의 한 상인 바테라이트(vaterite) 결정형의 탄산칼슘 입자가 형성되었음을 확인할 수 있었다.Further, as shown in FIGS. 5C and 5D, the calcium carbonate element component was confirmed by EDX (energy-dispersive X-ray spectroscopy) analysis of the PEG-modified keratin nanoparticles. FT-IR (Fourier Transform Infrared) It was confirmed that calcium carbonate particles of vaterite crystal type, which is one of calcium, were formed.

실험예Experimental Example 2: 케라틴의  2: of keratin UGT1A1의UGT1A1 mRNAmRNA 발현  Expression 상승 효과Synergistic effect (실시간  (real time qPCRqPCR ))

실시예 1에서 제조한 케라틴을 간세포(hepatocyte)에 처리하고, mRNA의 발현을 실시간 qPCR로 확인하였다. The keratin prepared in Example 1 was treated with hepatocytes and the expression of mRNA was confirmed by real-time qPCR.

구체적으로 1x104 cells/cm2 HepG2 세포를 6웰 플레이트에 접종하고 24시간동안 인큐베이션한 후, 일반배지, 0.5 % (w/v) 케라틴 배지로 교체하였다. 48시간동안 인큐베이션한 후, 배지를 제거하고 DPBS로 세척해 주었다. RNA를 추출하기 위해 하이브리드-R 키트(Gene All)를 사용하였고, 나노드롭(MICROP UV-Vis Spectrophotometer M600)을 사용하여 정량하였다. Specifically, 1 × 10 4 cells / cm 2 HepG2 cells were inoculated into 6-well plates, incubated for 24 hours, and then replaced with normal medium and 0.5% (w / v) keratin medium. After incubation for 48 hours, the medium was removed and washed with DPBS. Hybrid-R kit (Gene All) was used to extract RNA and quantified using nano drop (MICROP UV-Vis Spectrophotometer M600).

1 ㎍의 RNA를 AccuPowerrⓡ Cycle Script RT Premix (Bioneer, 대전, 한국)에 첨가해서 열순환기(T106, Bio-Rad)를 이용해 cDNA를 만들었다. 사용한 프라이머의 서열은 하기 [표 1]과 같다.1 ㎍ of RNA was added to the AccuPowerr ⓡ Cycle Script RT Premix (Bioneer , Daejeon, Korea) made by the cDNA using a thermal cycler (T106, Bio-Rad). The sequences of primers used are shown in Table 1 below.

sensesense antisenseantisense UGT1A1 UGT1A1 5'-GAG AGA GGT GAC TGT CCA GGA C-3'5'-GAG AGA GGT GAC TGT CCA GGA C-3 ' 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3'5'-CAA ATT CCT GGG ATA GTG GAT TTT-3 ' β-Actin β-Actin 5'-GTC AGG CAG CTC GTA GCT CT-3'5'-GTC AGG CAG CTC GTA GCT CT-3 ' 5'-TCG TGC GTG ACA TTA AGG AG-3'5'-TCG TGC GTG ACA TTA AGG AG-3 '

각각의 반응에 SYBR Green PCR Mix 10μL, 0.5 pmole의 프라이머(sense와 antisense)와 50 nmole의 주형(template)을 넣어주었다. RT-PCR은 RG6000 5plex HRM (Corbett Research)기기를 사용하였으며, 95 ℃에서 15초, 어닐링(annealing) 온도 57 ℃에서 45초로, 40 사이클을 설정하였다. 역치주기(Threshold cycle, Ct) 값을 측정하기 위해, 상대정량(comparative Ct (2-ΔΔCt))법을 사용하였다. For each reaction, 10 μL of SYBR Green PCR Mix, 0.5 pmole of primer (sense and antisense) and 50 nmole template were added. RT-PCR was carried out using RG6000 5plex HRM (Corbett Research) instrument and set for 40 cycles at 95 ° C for 15 seconds and annealing temperature of 57 ° C for 45 seconds. To measure the threshold cycle (Ct) value, the relative Ct (2 -ΔΔCt ) method was used.

그 결과, 도 6에서 확인할 수 있는 바와 같이 대조군(케라틴 비처리군)에 비하여 케라틴 처리군은 UGT1A1의 mRNA 발현을 현저히 상승시켰다.As a result, as shown in FIG. 6, the keratin-treated group significantly increased the mRNA expression of UGT1A1 as compared with the control group (non-keratinized group).

실험예Experimental Example 3: 케라틴의  3: of keratin UGT1A1의UGT1A1 분자 발현  Molecular expression 상승 효과Synergistic effect (면역세포화학 염색) (Immunocytochemical staining)

실시예 1에서 제조한 케라틴을 간세포(hepatocyte)에 처리하고, 분자 발현을 면역염색세포화학 염색법으로 확인하였다. The keratin prepared in Example 1 was treated with hepatocytes and the molecular expression was confirmed by immunohistochemical staining.

구체적으로, 1x104 cells/cm2 HepG2 세포를 6웰 플레이트에 접종하고 24시간동안 인큐베이션한 후, 일반배지, 0.5 % (w/v) 케라틴 배지로 교체하였다. 48시간동안 인큐베이션한 후, 배지를 제거하고 DPBS로 세척해 주었다. 세척 후, 4% 파라포름알데하이드를 10분간 처리하여 고정시킨 후 DPBS로 세척해 주었다. 고정 후 0.1% triton X-100를 30분간 처리하여 permeabilization 시킨 후, 10% (w/v) normal goat serum을 1시간 동안 처리하였으며, 1:200으로 희석된 rabbit anti-human UGT1A1 antibody와 mouse anti-human integrin beta1 antibody를 처리하여 24시간동안 4℃에서 반응시켰다. 24 시간 반응 후, DPBS로 세 번 세척해 주었으며, secondary Alexa Fluor 546 conjugated antibody 와 FITC-conjugated antibody를 상온에서 1시간동안 처리하였다. 1시간 처리후 DPBS로 세 번 세척해 주었으며, 4',6-디아미디노-2-페닐인돌(DAPI)를 처리하였다. 염색된 세포들을 형광현미경(Olympus IX71)으로 관찰하였다.Specifically, 1 × 10 4 cells / cm 2 HepG2 cells were inoculated into 6-well plates, incubated for 24 hours, and then replaced with normal medium and 0.5% (w / v) keratin medium. After incubation for 48 hours, the medium was removed and washed with DPBS. After washing, 4% paraformaldehyde was fixed for 10 minutes and washed with DPBS. After incubation with 0.1% triton X-100 for 30 minutes, the cells were permeabilized and treated with 10% (w / v) normal goat serum for 1 hour. Rabbit anti-human UGT1A1 antibody diluted 1: 200 and mouse anti- human integrin beta1 antibody and reacted at 4 ° C for 24 hours. After 24 hours of reaction, the cells were washed three times with DPBS, and secondary Alexa Fluor 546 conjugated antibody and FITC-conjugated antibody were treated at room temperature for 1 hour. After 1 hour of treatment, it was washed three times with DPBS and treated with 4 ', 6-diamidino-2-phenylindole (DAPI). The stained cells were observed with a fluorescence microscope (Olympus IX71).

그 결과, 도 7에서 확인할 수 있는 바와 같이 대조군에 비하여 케라틴 처리군은 UGT1A1의 분자 발현을 현저하게 상승시켰다. As a result, as shown in Fig. 7, the keratin-treated group significantly increased the molecular expression of UGT1A1 as compared with the control group.

실험예Experimental Example 4: 케라틴의 막 수용체 및  4: membrane receptor of keratin and 시토졸Cetosol 수용체의 발현 조절 (실시간  Regulation of receptor expression qPCRqPCR ))

케라틴 매개성 UGT1A1 유도의 메커니즘을 연구하기 위하여, 다양한 막 수용체 및 시토졸 수용체의 발현을 실시간 qPCR로 평가하였다. In order to study the mechanism of keratin-mediated UGT1A1 induction, the expression of various membrane receptors and cytosolic receptors was evaluated by real-time qPCR.

구체적으로 실험예 2와 동일한 방법으로 UTG1A1의 mRNA 발현과 함께 시토졸 수용체인 GR (Glucocorticoid Receptor), 및 AhR (Aryl Hydrocarbon Receptor)의 mRNA의 발현을 확인하였다. 사용한 프라이머의 서열은 하기 [표 2]와 같다.Specifically, mRNA expression of cytosolic receptors GR (Glucocorticoid Receptor) and AhR (Aryl Hydrocarbon Receptor) was confirmed in the same manner as Experimental Example 2 with the expression of UTG1A1 mRNA. The sequences of the primers used are shown in Table 2 below.

sensesense antisenseantisense UGT1A1 UGT1A1 5'-GAG AGA GGT GAC TGT CCA GGA C-3'5'-GAG AGA GGT GAC TGT CCA GGA C-3 ' 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3'5'-CAA ATT CCT GGG ATA GTG GAT TTT-3 ' β-Actin β-Actin 5'-GTC AGG CAG CTC GTA GCT CT-3'5'-GTC AGG CAG CTC GTA GCT CT-3 ' 5'-TCG TGC GTG ACA TTA AGG AG-3'5'-TCG TGC GTG ACA TTA AGG AG-3 ' GRGR 5'-CT AAT GGC TAT TCA AGC CCC AGC AT-3'5'-CT AAT GGC TAT TCA AGC CCC AGC AT-3 ' 5'-TGA TTT CAG CTA ACA TCT CGG GGA ATT CAA T-3'5'-TGA TTT CAG CTA ACA TCT CGG GGA ATT CAA T-3 ' AhRAhr 5'-TG GTC TCC CCC AGA CAG TAG-3'5'-TG GTC TCC CCC AGA CAG TAG-3 ' 5'-TTC ATT GCC AGA AAA CCA GA-3'5'-TTC ATT GCC AGA AAA CCA GA-3 '

그 결과, 도 8에서 확인할 수 있는 바와 같이, 대조군에 비하여 케라틴을 처리한 경우, UGT1A1 뿐아니라, AhR 및 GR의 발현 역시 현저하게 그 발현을 증가시킴을 알 수 있었다. As a result, as can be seen from FIG. 8, when keratin was treated with respect to the control group, expression of not only UGT1A1 but also AhR and GR was remarkably increased.

실험예Experimental Example 5:  5: PEG화된PEGylated 케라틴의  Keratin UGT1A1의UGT1A1 mRNAmRNA 발현  Expression 상승 효과Synergistic effect (실시간  (real time qPCRqPCR ))

1x104 세포/cm2 HepG2 세포를 6 웰 플레이트에 접종하고 24시간 동안 인큐베이션한 후, 정상 배지, 0.5% 및 0.75% (w/v) PEG-g-케라틴 배지로 바꿔주었다. 12시간, 24시간 또는 48시간 동안 인큐베이션한 후, 배지를 제거하고 DPBS로 세척해 주었다. RNA를 추출하기 위해 Hybrid-R kit (Gene All)를 사용하였고, nanodrop (MICROP UV-Vis Spectrophotometer M600)을 사용하여 정량하였다. 1x10 4 cells / cm 2 HepG2 cells were inoculated into 6 well plates and incubated for 24 hours before being replaced with normal medium, 0.5% and 0.75% (w / v) PEG-g-keratin medium. After 12 hours, 24 hours or 48 hours of incubation, the medium was removed and washed with DPBS. Hybrid-R kit (Gene All) was used to extract RNA and quantified using nanodrop (MICROP UV-Vis Spectrophotometer M600).

1 ㎍의 RNA를 AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Korea)에 첨가해서 T106 Thermal Cycle (Bio-Rad)기기를 이용해 cDNA를 만들었다. UGT1A1프라이머는 5'-GAG AGA GGT GAC TGT CCA GGA C-3' (sense)와 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3' (antisense)이고, β-Actin 프라이머는 5'-GTC AGG CAG CTC GTA GCT CT-3' (sense)와 5'-TCG TGC GTG ACA TTA AGG AG-3' (antisense)를 사용하였다. 1 의 of RNA was added to AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Korea) and cDNA was prepared using T106 Thermal Cycle (Bio-Rad) instrument. The UGT1A1 primer is 5'-GAG AGA GGT GAC TGT CCA GGA C-3 '(sense) and 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3' (antisense) CAG CTC GTA GCT CT-3 '(sense) and 5'-TCG TGC GTG ACA TTA AGG AG-3' (antisense) were used.

각각의 반응은 SYBR Green PCR Mix 10μL, 0.5 pmole의 프라이머(sense와 antisense)와 50 nmole의 template를 넣어준다. 실시간 PCR은 RG6000 5plex HRM (Corbett Research)기기를 사용하였으며, 95℃에서 15초, 어닐링 온도 57℃에서 45초로 40 cycles로 설정하였다. Threshold cycle (Ct) 값을 측정하기 위해, comparative Ct (2-△△Ct) 방법을 사용하였다. For each reaction, add 10 μL of SYBR Green PCR Mix, 0.5 pmole of primer (sense and antisense) and 50 nmole template. Real-time PCR was performed using a RG6000 5plex HRM (Corbett Research) instrument and set to 40 cycles at 95 ° C for 15 seconds and annealing temperature 57 ° C for 45 seconds. To measure the threshold cycle (Ct) value, the comparative Ct (2 - ΔΔCt ) method was used.

그 결과, 도 9에서 확인할 수 있는 바와 같이 대조군(control)에 비하여, PEG화된 케라틴(PEG-Keratin)은 UGT1A1 mRNA의 발현을 증가시켰으며, 특히 PEG화된 케라틴 처리 48시간 후, UGT1A1의 mRNA 발현은 현저하게 증가함을 확인하였다. As a result, as shown in FIG. 9, PEG-keratin increased the expression of UGT1A1 mRNA, especially after 48 hours of PEGylated keratin treatment, mRNA expression of UGT1A1 .

실험예Experimental Example 6:  6: PEG화된PEGylated 케라틴의  Keratin UGT1A1UGT1A1 관련  relation 시토졸Cetosol 수용체의 발현 증가 (실시간  Increased receptor expression (real time qPCRqPCR ))

1x104 세포/cm2 HepG2 세포를 6웰 플레이트에 접종하고 24시간 동안 인큐베이션한 후, 정상 배지, 0.5% 또는 1.0% (w/v) PEG-g-케라틴 배지로 바꿔주었다. 12시간, 24시간, 36시간 또는 48시간 동안 인큐베이션한 후, 배지를 제거하고 DPBS로 세척해 주었다. RNA를 추출하기 위해 Hybrid-R kit (Gene All)를 사용하였고, nanodrop (MICROP UV-Vis Spectrophotometer M600)을 사용하여 정량하였다. 1x10 4 cells / cm 2 HepG2 cells were inoculated into 6 well plates and incubated for 24 hours and then replaced with normal medium, 0.5% or 1.0% (w / v) PEG-g-keratin medium. After 12, 24, 36 or 48 hours of incubation, the medium was removed and washed with DPBS. Hybrid-R kit (Gene All) was used to extract RNA and quantified using nanodrop (MICROP UV-Vis Spectrophotometer M600).

1 ㎍의 RNA를 AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Korea)에 첨가해서 T106 Thermal Cycle (Bio-Rad)기기를 이용해 cDNA를 만들었다. UGT1A1 프라이머는 5'-GAG AGA GGT GAC TGT CCA GGA C-3' (sense)와 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3' (antisense)이고, Constitutive Androstane Receptor (CAR)은 5'-TGA TCA GCT GCA AGA GGA GA-3' (sense)와 5'-AGG CCT AGC AAC TTC GCA TA-3'(antisense), 글루코코르티코이드 수용체(Glucocorticoid Receptor, GR) 프라이머는 5'-CT AAT GGC TAT TCA AGC CCC AGC AT-3'(sense)와 5'-TGA TTT CAG CTA ACA TCT CGG GGA ATT CAA T-3'(antisense), 아릴 하이드로카본 수용체(Aryl Hydrocarbon Receptor, AhR) 프라이머는 5'-TG GTC TCC CCC AGA CAG TAG-3' (sense), 5'-TTC ATT GCC AGA AAA CCA GA-3'(antisense)를 사용하였다. 또한 β-Actin 프라이머는 5'-GTC AGG CAG CTC GTA GCT CT-3' (sense)와 5'-TCG TGC GTG ACA TTA AGG AG-3' (antisense)를 사용하였다. 1 의 of RNA was added to AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Korea) and cDNA was prepared using T106 Thermal Cycle (Bio-Rad) instrument. The UGT1A1 primer is 5'-GAG AGA GGT GAC TGT CCA GGA C-3 '(sense) and 5'-CAA ATT CCT GGG ATA GTG GAT TTT-3' (antisense) and Constitutive Androstane Receptor (CAR) TGA TCA GCT GCA AGA GGA GA-3 '(sense) and 5'-AGG CCT AGC AAC TTC GCA TA-3' (antisense), Glucocorticoid receptor (GR) primers are 5'-CT AAT GGC TAT TCA AGC CCC AGC AT-3 '(sense) and 5'-TGA TTT CAG CTA ACA TCT CGG GGA ATT CAA T-3' (antisense), Aryl Hydrocarbon Receptor (AhR) TCC CCC AGA CAG TAG-3 '(sense) and 5'-TTC ATT GCC AGA AAA CCA GA-3' (antisense). In addition, 5'-GTC AGG CAG CTC GTA GCT CT-3 '(sense) and 5'-TCG TGC GTG ACA TTA AGG AG-3' (antisense) were used as β-Actin primers.

각각의 반응은 SYBR Green PCR Mix 10 μL, 0.5 pmole의 프라이머(sense와 antisense)와 50 nmole의 template를 넣어준다. 실시간 PCR은 RG6000 5plex HRM (Corbett Research)기기를 사용하였으며, 95℃에서 15초, 어닐링 온도 57℃에서 45초로 40 cycles로 설정하였다. Threshold cycle (Ct) 값을 측정하기 위해, comparative Ct (2-△△Ct) 방법을 사용하였다. For each reaction, add 10 μL of SYBR Green PCR Mix, 0.5 pmole of primer (sense and antisense) and 50 nmole template. Real-time PCR was performed using a RG6000 5plex HRM (Corbett Research) instrument and set to 40 cycles at 95 ° C for 15 seconds and annealing temperature 57 ° C for 45 seconds. To measure the threshold cycle (Ct) value, the comparative Ct (2 - ΔΔCt ) method was used.

그 결과, 도 10에서 확인할 수 있는 바와 같이 대조군(control)에 비하여, PEG화된 케라틴(PEG-Keratin)은 UGT1A1 및 관련 시토졸 수용체(AhR, CAR 및 GR의 발현을 증가시켰다. As a result, as shown in Fig. 10, PEG-keratin increased the expression of UGT1A1 and related cytosolic receptors (AhR, CAR and GR) as compared to the control.

실험예Experimental Example 7:  7: PEG화된PEGylated 케라틴의 간 보호 효과  Keratin's liver protection effect

PEG화된 케라틴의 간 보호 효과를 확인하기 위하여 6주령의 수컷 BALB/c계 마우스를 구입하여 실험에 사용하였다. 마우스는 1주간 동물 실험실에 순화시켜 그 기간 중 일반증상을 관찰하고 이상이 없는 동물만을 선택하여 실험에 사용하였다. 마우스는 실험군별로 각 4마리씩 사용하였다. 실험기간 중 사육실 환경조건은 실내온도 23℃, 상대습도는 50%, 조명시간 12시간(오전8시~오후 8시) 및 조도 200-300 Lux를 유지하였다. 모든 실험동물은 플라스틱 케이지에서 2마리씩 사육하였다. 사료는 마우스용 고형사료를, 물은 상수도를 자유롭게 섭취시켰다. 간 독성 유발을 위하여 20 (w/v) % 에탄올(EtOH)을 2주간 2.5 ml/kg씩 경구투여하고 그 후 2주간은 5.0 ml/kg씩 매일 경구투여하였다. A 6 - week - old male BALB / c mouse was purchased and used for the experiment to confirm the liver protective effect of PEGylated keratin. Mice were sanitized in an animal laboratory for 1 week and observed general symptoms during the period. Only animals with no abnormalities were selected for the experiment. Four mice were used for each experimental group. During the experimental period, the room temperature was maintained at 23 ℃, the relative humidity was 50%, the lighting time was 12 hours (8:00 am to 8:00 pm) and the illumination was 200-300 lux. All experimental animals were housed in plastic cages. Diets were fed a solid diet for mice and water was freely consumed. Twenty (w / v)% ethanol (EtOH) was orally administered at 2.5 ml / kg for 2 weeks to induce hepatotoxicity, and then administered orally daily at 5.0 ml / kg for 2 weeks.

또한, PEG화된 케라틴의 간 보호 효과를 확인하기 위하여 에탄올을 경구 투여하고 6시간 후, 1(w/v)% Pinitol(간독성 완화 효과가 있다고 알려짐), Amicogen Co. (Jinju, Korea), 또는 1(w/v)% PEG화된 케라틴을 2주간은 2.5 ml/kg씩 경구투여하고 2주간은 5.0 ml/kg씩 매일 경구투여하였다. 4주간의 사육 후, 마우스를 안락사시켰으며, 전체 간을 취득하였고 채혈하였다. 그리고 취득한 간의 무게를 측정하였으며, 채혈한 혈액의 생화학적 검사는 AST (aspartate amino transferase), ALT (alanine amino transferase), total cholesterol를 측정하기 위하여 자동혈액생화학분석기(200 FR, TOSIBA, Tokyo, Japan)를 사용하였다. 또한 채취한 간조직을 1.15 % KCl 용액으로 균질화 하여, 얻은 샘플을 원심분리 하였고, 균질화된 간조직의 total glutathione (GSH)을 Glutathione Assay Kit (Sigma CS0260)를 이용하여 제조사의 사용방법에 따라 측정하였다.To confirm the liver protection effect of PEGylated keratin, ethanol was orally administered and after 6 hours, 1 (w / v)% Pinitol (known to have a hepatotoxic effect), Amicogen Co. (Kangnam, Korea) or 1 (w / v)% PEGylated keratin was orally administered at 2.5 ml / kg for 2 weeks and 5.0 ml / kg for 2 weeks. After 4 weeks of breeding, the mice were euthanized and whole liver was obtained and blood was collected. The biochemical tests of blood samples were performed using an automatic blood biochemical analyzer (200 FR, TOSIBA, Tokyo, Japan) to measure AST (aspartate amino transferase), alanine aminotransferase (ALT), and total cholesterol. Were used. The obtained liver tissue was homogenized with 1.15% KCl solution, and the obtained sample was centrifuged. The total glutathione (GSH) of the homogenized liver tissue was measured according to the manufacturer's method using Glutathione Assay Kit (Sigma CS0260) .

그 결과, 도 11에서 확인할 수 있는 바와 같이 간의 무게에서는 유의한 차이가 없었으나, 에탄올로 유발된 간 손상, 독성에 의해 높아진 AST (aspartate amino transferase) 및 ALT (alanine amino transferase) 효소들의 활성도가 PEG화된 케라틴 또는 Pinitol의 투여군에서 낮아지는 것이 관찰되었다. 또한 에탄올로 유발된 간 손상, 독성에 의해 높아진 혈중 콜레스테롤의 농도가 PEG화된 케라틴 또는 Pinitol의 투여군에서 낮아지는 것이 관찰되었다. 나아가, 간 손상시 저하되는 글루타티온(GSH)의 농도가 PEG화된 케라틴과 Pinitol의 투여군에서 정상치로 회복되는 것을 관찰할 수 있었다. As a result, as shown in FIG. 11, there was no significant difference in liver weight, but the activity of ethanol-induced liver injury, aspartate amino transferase (ALT) and alanine amino transferase And decreased in the keratin or Pinitol administration group. In addition, ethanol - induced liver damage, increased blood cholesterol concentration due to toxicity was observed to be lower in PEGylated keratin or Pinitol treated group. Furthermore, it was observed that the concentration of glutathione (GSH), which is lowered in liver injury, is restored to the normal level in the PEGylated keratin and Pinitol group.

<110> University-Industry Cooperation Group of Kyung Hee University AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs, method for preparing the same and transporter carrying and releasing drugs using the complex nanoparticle <130> P15-181-KHU <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for UGT1A1 <400> 1 gagagaggtg actgtccagg ac 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for UGT1A1 <400> 2 caaattcctg ggatagtgga tttt 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for beta-Actin <400> 3 gtcaggcagc tcgtagctct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for beta-Actin <400> 4 tcgtgcgtga cattaaggag 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Glucocorticoid Receptor <400> 5 ctaatggcta ttcaagcccc agcat 25 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for Glucocorticoid Receptor <400> 6 tgatttcagc taacatctcg gggaattcaa t 31 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Aryl Hydrocarbon Receptor <400> 7 tggtctcccc cagacagtag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for Aryl Hydrocarbon Receptor <400> 8 ttcattgcca gaaaaccaga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Constitutive Androstane Receptor <400> 9 tgatcagctg caagaggaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for Constitutive Androstane Receptor <400> 10 aggcctagca acttcgcata 20 <110> University-Industry Cooperation Group of Kyung Hee University          AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Complex nanoparticle comprising PEGylated keratin with relieving          side effects of drugs, method for preparing the same and          transporter carrying and releasing drugs using the complex          nanoparticle <130> P15-181-KHU <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for UGT1A1 <400> 1 gagagaggtg actgtccagg ac 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for UGT1A1 <400> 2 caaattcctg ggatagtgga tttt 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for beta-Actin <400> 3 gtcaggcagc tcgtagctct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for beta-Actin <400> 4 tcgtgcgtga cattaaggag 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Glucocorticoid Receptor <400> 5 ctaatggcta ttcaagcccc agcat 25 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for Glucocorticoid Receptor <400> 6 tgatttcagc taacatctcg gggaattcaa t 31 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Aryl Hydrocarbon Receptor <400> 7 tggtctcccc cagacagtag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for Aryl Hydrocarbon Receptor <400> 8 ttcattgcca gaaaaccaga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for Constitutive Androstane Receptor <400> 9 tgatcagctg caagaggaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for Constitutive Androstane Receptor <400> 10 aggcctagca acttcgcata 20

Claims (20)

케라틴을 유효성분으로 포함하는 간 보호용 약학적 조성물. A pharmaceutical composition for liver protection comprising keratin as an active ingredient. 제1항에 있어서, 상기 케라틴은 머리카락 유래의 케라틴인 약학적 조성물. The pharmaceutical composition according to claim 1, wherein the keratin is a hair-derived keratin. 제1항에 있어서, 상기 케라틴은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴인 약학적 조성물. The pharmaceutical composition according to claim 1, wherein the keratin is a polyethylene glycol (PEGylated) keratin. 제3항에 있어서, 상기 PEG화된 케라틴은 케라틴의 아민기에 PEG가 결합된 것인 약학적 조성물. 4. The pharmaceutical composition of claim 3, wherein the PEGylated keratin is PEG bound to the amine group of the keratin. 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간 보호용 약학적 조성물. A pharmaceutical composition for liver protection comprising, as an active ingredient, polyethylene glycol (PEGylated) keratin nanoparticles. 제5항에 있어서, 상기 PEG화된 케라틴은 케라틴의 아민기에 PEG가 결합된 것인 약학적 조성물. 6. The pharmaceutical composition according to claim 5, wherein the PEGylated keratin is PEG bound to an amine group of the keratin. 제5항에 있어서, 상기 PEG화된 케라틴 나노입자는 PEG화된 케라틴을 중성 수계 용액에 0.2 %(w/v) 이상 첨가하여 제조한 것인 약학적 조성물. 6. The pharmaceutical composition according to claim 5, wherein the PEGylated keratin nanoparticles are prepared by adding PEGylated keratin to a neutral aqueous solution by at least 0.2% (w / v). 제5항에 있어서, 상기 PEG화된 케라틴 나노입자는 고빌리루빈 혈증, 빌리루빈 유도성 뇌기능 장애, 용혈성 황달 및 간독성으로 이루어진 군에서 선택되는 어느 하나 이상을 부작용으로 갖는 약물을 담지하는 것인 약학적 조성물. The pharmaceutical composition according to claim 5, wherein the PEGylated keratin nanoparticle carries a drug having at least one of side effects selected from the group consisting of hyperbilirubinemia, bilirubin induced brain dysfunction, hemolytic jaundice and hepatotoxicity. . 제8항에 있어서, 상기 약물은 아타자나비어(atazanavir), 세프트리악손(ceftriaxone), 아세트아미노펜(acetaminophen), 벨리노스타트(belinostat), 이리노테칸(irinotecan), 세팔로스포린(cephalosporin), 답손(dapsone), 레보도파(levodopa), 레보플록사신(levofloxacin), 메틸도파(methyldopa), 니트로푸란토인(nitrofurantoin), 페니실린(penicillin), 바클로펜(baclofen), 클로나제팜(clonazepam), 트리헥실페니딜(trihexylphenidyl), 부프레노르핀(buprenorphine), 카르베딜롤(carvedilol), 독소루비신(doxorubicin), 에티닐에스트라디올(ethinylestradiol), 엔타카폰(entacapone), SN-38, 에토포시드(etoposide), 에제티미브(ezetimibe), 몰핀(morphine), 날롤핀(nalorphine), 날트렉손(naltrexone), 레티가빈(retigabine), 트로포테칸(tropotecan) 및 트로글리타존(troglitazone)으로 이루어진 군에서 선택되는 어느 하나 이상인 약학적 조성물. 9. The method of claim 8, wherein the drug is selected from the group consisting of atazanavir, ceftriaxone, acetaminophen, belinostat, irinotecan, cephalosporin, dapsone, levodopa, levofloxacin, methyldopa, nitrofurantoin, penicillin, baclofen, clonazepam, trihexylpyridyl (e.g., trihexylphenidyl), buprenorphine, carvedilol, doxorubicin, ethinylestradiol, entacapone, SN-38, etoposide, A pharmaceutical composition comprising at least one selected from the group consisting of ezetimibe, morphine, nalorphine, naltrexone, retigabine, tropotecan and troglitazone. . 케라틴을 유효성분으로 포함하는 간 보호용 건강기능식품 조성물.A health functional food composition for liver protection comprising keratin as an active ingredient. 제10항에 있어서, 상기 케라틴은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴인 건강기능식품 조성물. 11. The composition of claim 10, wherein the keratin is a polyethylene glycol (PEGylated) keratin. 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간 보호용 건강기능식품 조성물. A health functional food composition for liver protection comprising an active ingredient of polyethylene glycol (PEGylated) keratin nanoparticles. 케라틴을 유효성분으로 포함하는 간독성 질환 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for the prevention or treatment of hepatotoxic diseases comprising keratin as an active ingredient. 제13항에 있어서, 상기 케라틴은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴인 약학적 조성물. 14. The pharmaceutical composition according to claim 13, wherein the keratin is a polyethylene glycol (PEGylated) keratin. 제13항 또는 제14항에 있어서, 상기 간독성 질환은 약물성 간 손상, 바이러스성 간 손상, 간염, 간경화, 간암 또는 간성혼수인 약학적 조성물. 15. The pharmaceutical composition according to claim 13 or 14, wherein the hepatotoxic disease is drug substance damage, viral liver damage, hepatitis, liver cirrhosis, liver cancer or hepatic coma. 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간독성 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of hepatotoxic diseases comprising, as an active ingredient, polyethylene glycol (PEGylated) keratin nanoparticles. 제16항에 있어서, 상기 간독성 질환은 약물성 간 손상, 바이러스성 간 손상, 간염, 간경화, 간암 또는 간성혼수인 약학적 조성물. 17. The pharmaceutical composition according to claim 16, wherein the hepatotoxic disease is drug substance damage, viral liver damage, hepatitis, liver cirrhosis, liver cancer or hepatic coma. 케라틴을 유효성분으로 포함하는 간독성 질환 예방 또는 개선용 건강기능식품 조성물. A health functional food composition for preventing or ameliorating hepatotoxic diseases comprising keratin as an active ingredient. 제18항에 있어서, 상기 케라틴은 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴인 건강기능식품 조성물. The health functional food composition according to claim 18, wherein the keratin is polyethylene glycol (PEG) -ated keratin. 폴리에틸렌글리콜(polyethylene glycol, PEG)화된(PEGylated) 케라틴 나노입자를 유효성분으로 포함하는 간독성 질환 예방 또는 개선용 건강기능식품 조성물. A health functional food composition for preventing or ameliorating hepatotoxic diseases comprising, as an active ingredient, polyethylene glycol (PEGylated) keratin nanoparticles.
KR1020160099228A 2016-08-04 2016-08-04 Complex nanoparticle comprising PEGylated keratin with relieving side effects of drugs, method for preparing the same and transporter carrying and releasing drugs using the complex nanoparticle KR20180015807A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110063945A (en) * 2019-04-15 2019-07-30 温州医科大学 A kind of bilirubin nano particle and preparation method thereof for treating acute pancreatitis
CN111972399A (en) * 2020-08-06 2020-11-24 温州医科大学 Preservation solution for maintaining activity of liver cells

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110063945A (en) * 2019-04-15 2019-07-30 温州医科大学 A kind of bilirubin nano particle and preparation method thereof for treating acute pancreatitis
CN110063945B (en) * 2019-04-15 2021-07-09 温州医科大学 Bilirubin nanoparticles for treating acute pancreatitis and preparation method thereof
CN111972399A (en) * 2020-08-06 2020-11-24 温州医科大学 Preservation solution for maintaining activity of liver cells

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