[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

KR20110061881A - Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity - Google Patents

Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity Download PDF

Info

Publication number
KR20110061881A
KR20110061881A KR1020090118416A KR20090118416A KR20110061881A KR 20110061881 A KR20110061881 A KR 20110061881A KR 1020090118416 A KR1020090118416 A KR 1020090118416A KR 20090118416 A KR20090118416 A KR 20090118416A KR 20110061881 A KR20110061881 A KR 20110061881A
Authority
KR
South Korea
Prior art keywords
acid
polyethyleneimine
bile
bile acid
association
Prior art date
Application number
KR1020090118416A
Other languages
Korean (ko)
Other versions
KR101128108B1 (en
Inventor
정지훈
채수영
Original Assignee
성균관대학교산학협력단
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.)
Filing date
Publication date
Application filed by 성균관대학교산학협력단 filed Critical 성균관대학교산학협력단
Priority to KR1020090118416A priority Critical patent/KR101128108B1/en
Publication of KR20110061881A publication Critical patent/KR20110061881A/en
Application granted granted Critical
Publication of KR101128108B1 publication Critical patent/KR101128108B1/en

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/7105Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/711Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

PURPOSE: A conjugate containing polyethyleneimine and bile acid is provided to ensure high transport efficiency and to transport nucleic acid drugs. CONSTITUTION: A conjugate containing polyethyleneimine and bile acid is conjugated by reaction of carboxy group of bile acid and amine group of polyethyleneidmine. The bile acid is cholic acid, 5 beta-cholanic acid, chenodeoxycholic acid, glycoholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, or 7-oxo-lithochoilc acid. The conjugated contain macromolecule or hydrophobic drugs. The macromolecule is plasmid DNA, siRNA, or antisense oligonucleotide. The hydrophobic drug is doxorubicin, adriamycin, cisplatin, paclitaxel, taxol, docetaxel, or daunomycin.

Description

폴리에틸렌이민 및 담즙산으로 구성되는 세포막융합활성을 지닌 양친성 고분자 회합체{Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity}Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity}

본 발명은 양이온성 고분자와 담즙산 회합체를 이용한 핵산 또는 음이온성 단백질, 펩타이드 등의 거대분자와 음이온성 나노입자 등을 세포내로 전달할 수 있는 전달 시스템에 관한 것이다. 특히, 본 발명은 폴리에틸렌이민(polyethylenimine, PEI)와 담즙산을 함유하는 생체적합성 회합체에 관한 것으로, 1) 상기 담즙산이 PEI 골격에 직접 연결되거나 2) 연결체(crosslinker)를 통해 PEI골격에 연결된다. The present invention relates to a delivery system that can deliver macromolecules such as nucleic acids or anionic proteins, peptides and anionic nanoparticles using cationic polymers and bile acid conjugates into cells. In particular, the present invention relates to a biocompatible assembly containing polyethyleneimine (PEI) and bile acids, wherein 1) the bile acids are directly linked to the PEI backbone or 2) are linked to the PEI backbone via a crosslinker. .

DNA 및 단백질 같은 생물학적 거대분자(macromolecular)에 대한 세포 플라즈마 막(cell plasma membrane)의 제한된 투과성이 거대분자 약물의 치료학적 잠재력을 제한하는 강력한 장벽중에 하나이기 때문에, 수십년 동안 거대 분자의 세포막 투과를 촉진 시키는 전달 시스템(delivery systems)이 연구되어 왔다. 세포막의 물 리학적 투과성(예를 들어, electroporation), 리포좀을 이용한 막융합(membrane fusion)에 의한 직접 전달 및 엔도시토시스(endocytosis)라 불리는 콜로이드 입자의 에너지 의존성 세포내흡수(cellular uptake)와 같은 수 많은 연구가 진행되어 왔다. The limited permeability of cell plasma membranes to biological macromolecules, such as DNA and proteins, is one of the strong barriers to limiting the therapeutic potential of macromolecular drugs, which has led to cell membrane permeation of macromolecules for decades. Promoting delivery systems have been studied. Physical membrane permeability (eg electroporation), direct delivery by membrane fusion with liposomes, and energy dependent cellular uptake of colloidal particles called endocytosis. Many studies have been conducted.

최근에는, 막융합, 지질구조 재배열(lipid structure rearrangement), 또는 기공 형성(pore formation)을 통하여 막을 통과하게 하는 세포투과 펩타이드(cell penetrating peptides; CPPs)라고 불리는 일련의 양이온성 펩티드가 확인되었다(El-Andaloussi, S.; Holm, T.; Langel, U. Curr Pharm Des 2005, 11, 3597-611). 세포 투과 펩타이드의 막활성(membrane-active) 특성은 일반적으로 작은 분자, 단백질 및 핵산 약물의 세포내 전달에 사용된다. 비록 메커니즘이 명확히 규명되지는 않았으나, 펩타이드는 엔도시토시스 비의존적 및 엔도시토시스 의존적인 과정을 통하여 거대분자의 세포막 투과를 중재하는 것으로 판단된다(Plank, C.; Zauner, W.; Wagner, E. Adv Drug Deliv Rev 1998, 34, 21-35) Recently, a series of cationic peptides called cell penetrating peptides (CPPs) have been identified that allow membranes to pass through membrane fusion, lipid structure rearrangement, or pore formation. El-Andaloussi, S .; Holm, T .; Langel, U. Curr Pharm Des 2005, 11, 3597-611). The membrane-active properties of cell penetrating peptides are generally used for intracellular delivery of small molecules, proteins and nucleic acid drugs. Although the mechanism is not clearly identified, peptides are thought to mediate macromolecular membrane permeation of macromolecules through endocytosis-independent and endocytosis-dependent processes (Plank, C .; Zauner, W .; Wagner, E.). Adv Drug Deliv Rev 1998, 34, 21-35)

상기 펩타이드의 대부분은 양친매성(amphiphile) 특성과 전형적인 알파-헬릭스(α-helix)의 구조를 가지고 있으며, 친수성 아미노산은 펩타이드의 주된 축과 평행을 이루고 있다. 이러한 면상 양친매성 특성(facial amphiphilicity)은 막융합 또는 펩타이드의 투과성에 일정 역할을 하게 된다(Cheng, Y.; Ho, D. M.; Gottlieb, G. R.; Kahne, D.; Bruck, M. A. J Am Chem Soc 1992, 114, 7319-7320; Legendre, J. Y.; Szoka, F. C., Jr. Proc Natl Acad Sci USA 1993, 90, 893-7). Most of the peptides have amphiphile properties and typical alpha-helix structures, and the hydrophilic amino acids are parallel to the major axis of the peptide. Such amphiphilicity plays a role in membrane fusion or permeability of peptides (Cheng, Y .; Ho, DM; Gottlieb, GR; Kahne, D .; Bruck, MA J Am Chem Soc 1992, 114, 7319-7320; Legendre, JY; Szoka, FC, Jr. Proc Natl Acad Sci USA 1993, 90, 893-7).

데옥시콜린산(deoxycholic acid)과 같은 담즙산 계열은 특이한 구조적 특징을 가지며, 이는 2 내지 3개의 친수성 하이드록시기가 같은 방향을 향하고 있고 소수성 스테로이드 하이드로카본이 반대방향을 향하고 있는 것으로 설명할 수 있다(도 1참조)(Axelrod, H. R.; Sofia, M. J.; Walker, S. Adv Drug Deliv Rev 1998, 30, 61-71). 양친매성 담즙산은 지방 및 콜레스테롤의 용해제 및 전달제로 작용한다. 상기 담즙산 및 이들의 유도체는 막과 상호작용을 하여 극성 분자의 세포내흡수(cellular uptake)를 증진시키는 것으로 알려져 있다(Swenson, E. S.; Curatolo, W. J. Adv Drug Deliv Rev 1992, 8, 39-92; Walker, S.; Sofia, M. J.; Kakarla, R.; Kogan, N. A.; Wierichs, L.; Longley, C. B.; Bruker, K.; Axelrod, H. R.; Midha, S.; Babu, S.; Kahne, D. Proc Natl Acad Sci USA 1996, 93, 1585-90)Bile acid series, such as deoxycholic acid, have unusual structural characteristics, which can be explained by the fact that two to three hydrophilic hydroxyl groups are pointing in the same direction and the hydrophobic steroid hydrocarbons are pointing in the opposite direction (Fig. 1) (Axelrod, HR; Sofia, MJ; Walker, S. Adv Drug Deliv Rev 1998, 30, 61-71). Amphiphilic bile acids act as solubilizers and delivery agents for fats and cholesterol. The bile acids and their derivatives are known to interact with the membrane to enhance cellular uptake of polar molecules (Swenson, ES; Curatolo, WJ Adv Drug Deliv Rev 1992, 8, 39-92; Walker; , S .; Sofia, MJ; Kakarla, R .; Kogan, NA; Wierichs, L .; Longley, CB; Bruker, K .; Axelrod, HR; Midha, S .; Babu, S .; Kahne, D. Proc Natl Acad Sci USA 1996, 93, 1585-90)

이에 본 발명자는, 면상 양친매성 물질(facial amphiphile)인 담즙산 및 폴리에틸렌이민의 회합체를 연구한 결과, 효율적으로 거대분자를 세포내로 전달할 수 있음을 확인하고 본 발명을 완성하였다. The present inventors have studied the association of bile acids and polyethyleneimines, which are planar amphiphiles, and confirmed that the macromolecules can be efficiently delivered into cells, thereby completing the present invention.

본 발명은 세포내로 거대분자 등을 전달할 수 있는 전달 시스템을 제공하기 위한 것이다. The present invention is to provide a delivery system capable of delivering macromolecules and the like into the cell.

상기의 목적을 달성하기 위하여, 본 발명은 폴리에틸렌이민(polyethylenimine; PEI) 및 담즙산으로 구성된 회합체에 있어서, 상기 담즙산의 카르복시기와 폴리에틸렌이민의 아민기의 반응에 의하여 결합된 회합체를 제공한다. 특히, 상기 폴리에틸렌이민(polyethylenimine, PEI)와 담즙산을 함유하는 생체적합성 회합체에 관한 것으로 1) 상기 담즙산이 PEI 골격에 직접 연결되거나 2) 연결체(crosslinker)를 통해 PEI골격에 연결된다. In order to achieve the above object, the present invention provides a combined association by the reaction of the carboxyl group of the bile acid and the amine group of polyethyleneimine in the association consisting of polyethylenimine (PEI) and bile acid. In particular, the present invention relates to a biocompatible assembly containing the polyethylenimine (PEI) and bile acid. 1) The bile acid is directly connected to the PEI skeleton or 2) to the PEI skeleton through a crosslinker.

본 발명에서 사용되는 '폴리에틸렌이민'은, 단량체에 아민기가 있어 담즙산의 카르복시기와 반응할 수 있는 구조를 가지고 있다. 본 발명에서 사용되는 폴리에틸렌이민의 분자량은, 세포독성과 생체 내 투여 시 원활한 소실(clearance)을 고려할 때 100 내지 100kDa인 것이 바람직하다. 즉, 고분자량의 폴리에틸렌이민은 세포독성이 문제될 수 있고, 저분자량의 폴리에틸렌이민은 전달효율이 떨어지는 문제가 있어, 상기 분자량이 바람직하다.Polyethyleneimine used in the present invention has an amine group in the monomer, and has a structure capable of reacting with a carboxyl group of bile acids. The molecular weight of polyethyleneimine used in the present invention is preferably 100 to 100 kDa in consideration of cytotoxicity and smooth clearance during in vivo administration. That is, high molecular weight polyethyleneimine may have a problem of cytotoxicity, and low molecular weight polyethyleneimine has a problem of low transfer efficiency, so that the molecular weight is preferable.

본 발명에서 사용되는 '담즙산'은, 카르복시기가 폴리에틸렌이민의 아민기와 반응하여 아미드 결합을 하여 폴리에틸렌이민과 회합체(conjugate)를 형성할 수 있다. 담즙산으로는, 콜린산(cholic acid), 5β-콜란산(5β-cholanic acid), 체노데옥시콜린산(chenodeoxycholic acid), 글리코콜린산(glycocholic acid), 타우로콜린산(taurocholic acid), 데옥시콜린산(deoxycholic acid), 리쏘콜린산(lithocholic acid), 7-옥소-리소콜린산(7-oxo-lithocholic acid) 등을 포함하는 군으로부터 선택되는 담즙산이 바람직하게 사용될 수 있으며, 바람직하게는 콜린산, 데옥시콜린산, 리쏘콜린산을 사용할 수 있다. 'Bile acid' used in the present invention may be a carboxyl group reacts with an amine group of polyethyleneimine to form an conjugate with polyethyleneimine by amide bond. Bile acids include cholic acid, 5β-cholanic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, and taurocholic acid. A bile acid selected from the group comprising deoxycholic acid, lithocholic acid, 7-oxo-lithocholic acid, and the like may be preferably used. Cholic acid, deoxycholic acid and lithocholine acid can be used.

본 발명에 따른 담즙산과 폴리에틸렌이민으로 구성되는 회합체에서, 상기 담즙산과 상기 폴리에틸렌이민의 몰 비로 1 내지 50 % 포함되는 것이 바람직하며, 보다 바람직하게는 2 내지 15 %를 포함하는 것이 바람직하나 이에 한정하는 것은 아니다. 상기 몰 비란, 폴리에틸렌이민의 아민기의 전체 개수에 대하여 담즙산이 치환된 아민기의 개수를 의미하는 것으로, 이론상 몰 비가 100%란 폴리에틸렌이민의 아민기 모두에 담즙산이 치환된 것을 의미한다. In the assembly composed of bile acid and polyethyleneimine according to the present invention, it is preferable that the mole ratio of the bile acid and the polyethyleneimine is included in 1 to 50%, more preferably 2 to 15% but preferably limited thereto. It is not. The molar ratio means the number of amine groups in which bile acids are substituted with respect to the total number of amine groups of polyethyleneimine. In theory, the molar ratio of 100% means that bile acids are substituted in all of the amine groups of polyethyleneimine.

본 발명의 양이온성 담즙산 회합체는 수용액상에서 양친매성에 의해 자발적으로 미세 상분리를 일으켜 소수성 핵 및 친수성 껍질 구조의 규칙성을 나타내는 나노크기의 자기회합구조(self-assembled structure)를 형성하며, 임계미셀형성농도 가 0.001 내지 0.400 g/L 이고, 입자크기는 70 내지 500 nm로 나타난다. Cationic bile acid aggregates of the present invention spontaneously undergoes microphase separation by amphiphilicity in an aqueous solution to form nanoscale self-assembled structures exhibiting regularity of hydrophobic nuclei and hydrophilic shell structures, and critical micelles. Formation concentration is 0.001 to 0.400 g / L and particle size is 70 to 500 nm.

본 발명에 따른 담즙산과 폴리에틸렌이민으로 구성되는 회합체는 소수성 약물의 봉입 뿐만 아니라 핵산을 포함하는 음이온성 거대분자 또는 나노입자를 고이온복합체(polyelectrolyte complex) 나노입자 형태로 봉입하여 전달할 수 있다. 즉, 본 발명에 따른 회합체는 세포내로 잘 통과할 수 있으며, 이 때 거대분자 또는 소수성 약물을 봉입한 복합체 또한, 세포내로 통과할 수 있기 때문에 세포내로 전달하기 힘든 물질을 잘 전달할 수 있다. 상기 거대분자는 플라스미드 DNA, siRNA 및 안티센스올리고뉴클레오티드로 구성되는 군으로부터 선택될 수 있으며, 반드시 이에 한정되는 것은 아니다. The association consisting of bile acids and polyethyleneimine according to the present invention can be delivered by encapsulating hydrophobic drugs as well as anionic macromolecules or nanoparticles containing nucleic acids in the form of polyelectrolyte complex nanoparticles. That is, the assembly according to the present invention can pass well into the cell, and at this time, the complex encapsulated with the macromolecule or the hydrophobic drug can also pass through the cell, thereby delivering a substance that is difficult to deliver into the cell. The macromolecule may be selected from the group consisting of plasmid DNA, siRNA and antisense oligonucleotide, but is not necessarily limited thereto.

상기 소수성 약물로는 독소루비신(doxorubicin), 아드리아마이신(adriamycin), 시스플라틴(cisplatin), 파클리탁셀(paclitaxel) 또는 탁솔(taxol), 도세탁셀(docetaxel), 다우노마이신(daunomycin)등의 소수성 항암제와 조효소 Q10(coenzyme Q10), 비타민 E 및 그의 유도체, 비타민 A 및 그의 유도체등의 소수성 항산화제로 구성되는 군으로부터 선택될 수 있으며, 반드시 이에 한정되는 것은 아니다.The hydrophobic drugs include hydrophobic anticancer agents such as doxorubicin, adriamycin, adriamycin, cisplatin, paclitaxel or taxol, docetaxel, daunomycin and coenzyme Q10 ( coenzyme Q10), vitamin E and derivatives thereof, and vitamin A and derivatives thereof, and the like, and may be selected from the group consisting of hydrophobic antioxidants.

또한, 본 발명은 담즙산을 DCC 및 NHS로 활성화시키는 단계; 및 상기 활성된 담즙산에 폴리에틸렌이민을 첨가하는 단계를 포함하는 폴리에틸렌이민(PEI) 및 담즙산으로 구성된 회합체의 제조방법을 제공한다. In addition, the present invention comprises the steps of activating the bile acid with DCC and NHS; And it provides a method for producing an assembly consisting of polyethyleneimine (PEI) and bile acid comprising the step of adding polyethyleneimine to the activated bile acid.

본 발명에 따른 회합체 및 거대분자 또는 소수성 약물을 봉입한 복합체는 다음과 같은 특징이 있다. The complex and the complex containing the macromolecule or the hydrophobic drug according to the present invention are characterized as follows.

첫째, 종래 세포내로 거대분자를 전달하기 위해서 사용되는 물질들의 경우, 전달 효율이 좋지 못하고, 이의 제조를 위하여 복잡한 제조단계를 거쳐야 하는 경우가 많았다. 그러나 본 발명에 따른 경우, 간단한 방법으로 회합체를 제조할 수 있다. 또한 전달 효율이 매우 높아 특히 핵산약물을 세포내로 전달하는데 유용하게 사용될 수 있다. First, in the case of materials used to deliver macromolecules into conventional cells, the delivery efficiency is not good, and many cases have to go through complicated manufacturing steps to prepare them. However, in the case of the present invention, the assembly can be prepared by a simple method. In addition, the delivery efficiency is very high and may be particularly useful for delivering intracellular nucleic acid drugs.

둘째, 본 발명에 따른 회합체는 양친매성 특성을 가지고 있고, 이에 따라 거대분자를 효율적으로 전달할 수 있다. 또한 소수성 약물의 경우에도 양친매성 특성으로 인하여 효율적으로 봉입할 수 있어 약물 전달 시스템으로도 널리 활용할 수 있다. Second, the association according to the present invention has an amphiphilic property, and thus can efficiently transport macromolecules. In addition, even in the case of hydrophobic drugs can be efficiently encapsulated due to the amphipathic properties can be widely used as a drug delivery system.

이하,본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 더욱 쉽게 이해하기 위하여 제공되는 것일 뿐,실시예에 의하여 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be presented to assist in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

1. 사용 물질1. Material used

데옥시콜린산(Deoxycholic acid), 가지쇄 폴리에틸렌이민(branched polyethylenimine(PEI, Mw 25 kDa, 1.8 kDa)), 디사이클로헥실 카르보디이미드(dicyclohexyl carbodiimide; DCC), N-하이드록시 석신이미드(N-hydroxyl succinimide; NHS), 및 트리에틸아민(triethylamine ;TEA)은 시그마-알드리히사(Sigma-Aldrich (St. Louis, MO))로부터 구입하였다. 세포배양물질, DMEM(Dulbecco's modified Eagle's medium) 및 FBS(fetal bovine serum)은 깁코 비알엘사(Gibco BRL (Grand Island, NY))로부터 구입하였다. 엔도시토시스 억제제(chlorpromazine, wortmannin, genistein), 신진대사 억제제(sodium azide, 2-deoxy-glucose), 및 MTT(3-(4,5-dimethylthyazolyl-2)-2,5-diphenyl tetrazolium bromide)는 cell culture grade 제품이며, 시그마사(Sigma (St. Louis, MO))로부터 구입하였다. 나머지 다른 화학물질과 용매는 analytical grade 제품이며, 추가 정제없이 사용하였다. Deoxycholic acid, branched polyethylenimine (PEI, Mw 25 kDa, 1.8 kDa), dicyclohexyl carbodiimide (DCC), N-hydroxy succinimide (N -hydroxyl succinimide (NHS), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, Mo.). Cell culture material, Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). Endocytosis inhibitors (chlorpromazine, wortmannin, genistein), metabolic inhibitors (sodium azide, 2-deoxy-glucose), and MTT (3- (4,5-dimethylthyazolyl-2) -2,5-diphenyl tetrazolium bromide) Cell culture grade product, purchased from Sigma (Sigma (St. Louis, Mo.)). The other chemicals and solvents were analytical grade and used without further purification.

2. DA-PEI 회합체의 합성 및 특성2. Synthesis and Characterization of DA-PEI Associations

1) 회합체 합성1) association synthesis

도 1에 DA-PEI 회합체의 합성반응을 도시하였다. 1 shows the synthesis of DA-PEI aggregates.

먼저, 테트라하이드로퓨란(tetrahydrofuran)에 녹인 DA(deoxycholic acid) 1g (2.5 mmol)을 DCC(dicyclohexyl carbodiimide, 1.57 g, 7.6 mmol) 및 NHS(N- hydroxysuccinimide, 0.88 g, 7.6 mmol)으로 활성화시켰다(PLGA/DCC/NHS 몰비율=1:3:3). 활성된 DA를 n-헥산(n-hexane)에 침전시키고, 감압하여 건조시켰다. DA-PEI 회합체의 합성은 메틸렌클로라이드(methylenchloride)에서 활성화된 DA를 PEI 1.8 kDa에 첨가하여 수행하였다. DA1 및 DA3라 명명된 회합체를 제조하였으며, DA1의 몰 유입 비율(molar feed ratios)은 1:1(DA/PEI)이었고, DA3의 몰 유입 비율(molar feed ratios)은 1:3(DA/PEI)이었다. 생성물은 회전 증발장치로 건조시켰고, 0.1 M 염산에 녹인 후 아이스-콜드 아세톤/에테르(1:3 혼합)용액에서 침전시켰다. 침전물을 원심분리한 후, 건조-질소로 건조시켰다. 생성물은 증류수에 녹이고, 필터로 거른뒤, 동결건조하였다. 치환된 정도를 1H-NMR로 확인하였으며, 이를 도 2에 나타내었다. First, 1 g (2.5 mmol) of DA (deoxycholic acid) dissolved in tetrahydrofuran was activated with DCC (dicyclohexyl carbodiimide, 1.57 g, 7.6 mmol) and NHS (N-hydroxysuccinimide, 0.88 g, 7.6 mmol) (PLGA / DCC / NHS molar ratio = 1: 3: 3). Activated DA was precipitated in n-hexane and dried under reduced pressure. Synthesis of DA-PEI association was performed by adding DA activated in methylene chloride to 1.8 kDa of PEI. An assembly called DA1 and DA3 was prepared, the molar feed ratios of DA1 was 1: 1 (DA / PEI) and the molar feed ratios of DA3 was 1: 3 (DA / PEI). The product was dried by rotary evaporator, dissolved in 0.1 M hydrochloric acid and precipitated in ice-cold acetone / ether (1: 3 mixed) solution. The precipitate was centrifuged and then dried with dry-nitrogen. The product was dissolved in distilled water, filtered through a filter, and lyophilized. The degree of substitution was confirmed by 1 H-NMR, which is shown in FIG. 2.

2) 용혈성 시험(Hemolysis test)2) Hemolysis test

DA-PEI 회합체의 막 활성은 erythrocyte lysis assay로 측정하였다. SD rat로부터 배양된 혈액 700 g을 10분간 원심분리하였다. 펠렛(pellet)을 PBS로 3번 세척한 후 PBS에 재분산시켰다. Membrane activity of DA-PEI association was measured by erythrocyte lysis assay. 700 g of blood cultured from SD rats were centrifuged for 10 minutes. The pellet was washed three times with PBS and then redispersed in PBS.

용혈성 시험을 위하여, PBS에 용해된 적당량의 회합체를 1×108 RBC를 포함하는 용액에 첨가하였다. 시험 용액을 37℃ 에서 1 시간 배양한 후, 상기 용액을 최고 속도로 원심분리하였다. 헤모글로빈(hemoglobin)이 방출된 정도를 상청 액(supernatant)의 540 nm에서의 흡광도로 측정하였고, 이를 용혈 퍼센트로 표시하여 확인하였다. 용혈 퍼센트를 계산하기 위한 완전한 용혈은, 0.1 % Triton X-100를 첨가하여 수행하였고, 이를 대조군(100%)으로 사용하였다. For the hemolytic test, an appropriate amount of association dissolved in PBS was added to the solution containing 1 × 10 8 RBCs. After incubating the test solution at 37 ° C. for 1 hour, the solution was centrifuged at full speed. The degree of hemoglobin release was measured by absorbance at 540 nm of the supernatant, which was confirmed by expressing it in percent of hemolysis. Complete hemolysis to calculate the percent hemolysis was performed by adding 0.1% Triton X-100, which was used as a control (100%).

상기 회합체 합성 및 용혈성 시험의 결과는 하기 표 1과 같으며, 회합체에 포함된 담즙산의 양이 증가함에 따라 세포막활성이 증가됨을 관찰할 수 있다. 이에 세포막활성의 특징을 나타내며 용혈성이 적절하게 낮은 회합체를 선택하여 실험을 수행하였다. The results of the assembly synthesis and hemolytic test are shown in Table 1 below, and it can be observed that cell membrane activity increases as the amount of bile acid contained in the assembly increases. Therefore, experiments were performed by selecting associations exhibiting characteristics of cell membrane activity and having low hemolytic properties.

FRa FR a DSb DS b HLc HL c DA1DA1 1One 0.70.7 23.8%23.8% DA3DA3 33 2.52.5 32.9%32.9% a : 반응 유입비율(feed ratio; DA/PEI)
b : 1H-NMR로 확인된 치환 몰 비율(DA/PEI)
c : 20 ㎍/ml 농도의 murine erythrocyte에서의 용혈성
a: reaction feed ratio (DA / PEI)
b: substituted molar ratio (DA / PEI) as confirmed by 1 H-NMR
c: Hemolytic in murine erythrocyte at 20 ㎍ / ml

3. 고이온복합체(polyelectrolyte)의 형성과 특성3. Formation and Characterization of High Ion Polyelectrolytes

1) EMSA(Electrophoretic mobility shift assay)1) Electrophoretic mobility shift assay (EMSA)

회합체와 플라스미드 DNA간의 고이온복합체의 형성은 0.8 % agarose gel 에서 DNA 밴드의 EMSA로 확인되었다. 다양한 몰 비율의 DA-PEI 회합체와 0.3g의 플라스미드 DNA(pCMVLuc)를 PBS에 각각 희석시켰다. 희석된 회합체와 플라스미드 DNA 용액을 다른 질량비로 혼합하고, 잘 섞은 다음, 분석에 앞서 15분 동안 상온에 두었다. 전기영동(Electrophoresis)은 100 V에서 20분 동안 TAE 버퍼(40 mM Tris/HCl, 1 % (v/v) acetic acid, 1 mM EDTA)에서 수행되었다. 복합체의 이동 지연은 ethidium bromide staining으로 영상화하였다(도 3). The formation of high ionic complexes between the association and the plasmid DNA was confirmed by EMSA in the DNA band on 0.8% agarose gel. Various molar ratios of DA-PEI associations and 0.3 g of plasmid DNA (pCMVLuc) were each diluted in PBS. Diluted associations and plasmid DNA solutions were mixed at different mass ratios, mixed well and left at room temperature for 15 minutes prior to analysis. Electrophoresis was performed in TAE buffer (40 mM Tris / HCl, 1% (v / v) acetic acid, 1 mM EDTA) at 100 V for 20 minutes. The migration delay of the complex was imaged by ethidium bromide staining (FIG. 3).

도 3은, 합성된 회합체가 음이온성 거대분자인 플라스미드 DNA와의 이온 상호작용을 통해 고이온복합체 나노입자를 형성함으로써 아가로스겔 상에서의 이동성이 떨어지는 것을 나타낸다. Figure 3 shows that the synthesized aggregates lack mobility on agarose gel by forming high ionic complex nanoparticles through ionic interaction with plasmid DNA, which is an anionic macromolecule.

2) 크기 및 표면 전하 측정2) Size and surface charge measurements

DA-PEI 회합체와 플라스미드 DNA의 복합체에 대한 입도(hydrodynamic diameter) 및 표면 제타 전위(surface zeta potential)는 25℃에서 He-Ne 레이저를 구비한 동적 광산란 장치(dynamic light scattering device; Zeta Plus, Brookhaven Instrument Co., NY)를 이용하여 632 nm 에서 측정하였다. 모든 측정은 세 번 반복하였다. 측정된 크기 및 표면전하는 하기 표 2와 같다. The hydrodynamic diameter and surface zeta potential for the complex of DA-PEI associations and plasmid DNA were determined using a dynamic light scattering device (Zeta Plus, Brookhaven) with a He-Ne laser at 25 ° C. Instrument Co., NY) was used to measure at 632 nm. All measurements were repeated three times. The measured size and surface charge are shown in Table 2 below.

PSa PS a SCb SC b DA1DA1 79.8 nm79.8 nm +35.2 mV+35.2 mV DA3DA3 73.2 nm73.2 nm +33.2 mV+33.2 mV a : 복합체 크기(nm, 회합체/DNA 질량비 = 3)
b : 복합체 표면 전하(mV, 회합체/DNA 질량비 = 3)
a: complex size (nm, association / DNA mass ratio = 3)
b: complex surface charge (mV, aggregate / DNA mass ratio = 3)

4. 세포 배양, 트랜스팩션(transfection) 및 세포 생존률 측정(cell viability assays)4. Cell culture, transfection and cell viability assays

1) Transfection 및 reporter gene assay1) Transfection and reporter gene assay

인간 대장암 세포(human colorectal cancer cells (HCT116))를 10% FBS가 공급된 DMEM에서 배양하고, 37℃ 5% CO2의 조건을 유지하였다. Transfection을 위하여, 배양된 세포를 6-well plate에 접종한 후(2×105/well), 37℃ 5% CO2의 조건으로 24시간동안 배양하였다. 배양 배지를 혈청이 없는 배지로 교환한 후, 적당량의 폴리머/DNA 복합체를 첨가하였다. 4시간 배양후, transfection 배지를 제거하고, 10% FBS를 포함하는 성장배지(fresh growth medium)를 공급하였다. 상업적으로 널리 이용되는 luciferase assay kit(Promega, Madison, WI) 및 Lumat FB12 luminometer(Berthold GmbH, Pforzheim, Germany)로 transfection을 수행한 후 24시간 후에 루시퍼라아제(luciferase)의 활성을 측정하였다. BCA assay(Pierce, Rockford, IL)에 의하여 측정된 세포 단백질의 그램 당 발광정도(relative light unit; RLU)로 transfection의 효율을 나타내었다. 그 결과를 도 4에 나타내었다. 도 4에 나타난 바와 같이, DA3 회합체는 최적화된 조성의 PEI 25 kDa (w/w = 1)보다 10배 높은 transfection 효율을 나타내었다.Human colorectal cancer cells (HCT116) were incubated in DMEM fed with 10% FBS and maintained at 37 ° C. 5% CO 2 . For transfection, the cultured cells were inoculated into 6-well plates (2 × 10 5 / well) and then incubated for 24 hours under conditions of 5 ° C. CO 2 at 37 ° C. After replacing the culture medium with a serum-free medium, an appropriate amount of polymer / DNA complex was added. After 4 hours of incubation, the transfection medium was removed and a fresh growth medium containing 10% FBS was supplied. Luciferase activity was measured 24 hours after transfection with a commercially available luciferase assay kit (Promega, Madison, WI) and Lumat FB12 luminometer (Berthold GmbH, Pforzheim, Germany). The transfection efficiency was expressed by the relative light unit (RLU) per gram of cellular protein measured by BCA assay (Pierce, Rockford, IL). The results are shown in FIG. As shown in FIG. 4, the DA3 association showed a 10 times higher transfection efficiency than PEI 25 kDa (w / w = 1) of optimized composition.

2) Endocytosis and metabolic inhibitor studies2) Endocytosis and metabolic inhibitor studies

억제제의 첨가 및 reporter gene assay 전에 헤파린 설페이트(heparin sulfate)를 포함하는 PBS로 세척하는 단계를 제외하고는, transfection 방법을 상기 1)의 방법과 동일하게 수행하였다. The transfection method was performed in the same manner as in 1), except for the addition of the inhibitor and washing with PBS containing heparin sulfate before the reporter gene assay.

inhibitor assay를 위하여, 세포는 적절한 농도의 다양한 엔도시토시스 억제제(10 μg/ml chlorpromazine, 100 nM wortmannin, 200 μM genistein, 또는 상기 세 억제제의 조합) 및 대사억제제(0.1 % sodium azide 또는 50 mM 2-deoxy-glucose)의 존재하에 먼저 배양되었다. 다음으로, 폴리머/DNA 복합체를 동일한 억제제를 포함하는 혈청이 없는 배지 내의 세포에 첨가하고, 4시간 동안 배양한 후 10% 혈청을 포함하는 배지로 교체하였다. 24시간 더 배양한 후, 세포를 10 mg/ml 헤파린 설페이트(heparin sulfate)를 포함하는 PBS로 세척한 후, 다시금 PBS로 세척하였다. 세포 표면에 남아있는 복합체를 제거하기 위하여 추가적으로 헤파린 설페이트(heparin sulfate)로 세척하였다. 그 결과를 도 5에 나타내었다. For inhibitor assays, cells were treated with appropriate concentrations of various endocytosis inhibitors (10 μg / ml chlorpromazine, 100 nM wortmannin, 200 μM genistein, or a combination of the three inhibitors) and metabolic inhibitors (0.1% sodium azide or 50 mM 2- first incubated in the presence of deoxy-glucose). Next, the polymer / DNA complex was added to cells in a serum-free medium containing the same inhibitor, incubated for 4 hours and then replaced with a medium containing 10% serum. After 24 hours of incubation, the cells were washed with PBS containing 10 mg / ml heparin sulfate and then again with PBS. It was further washed with heparin sulfate to remove the complex remaining on the cell surface. The results are shown in Fig.

도 5에 도시된 바와 같이, 각각의 억제제 및 억제제의 조합 모두 DA3 회합체의 트랜스팩션에는 큰 영향을 미치지 않는 것으로 나타났다. 이는 DA3 회합체가 엔도시토시스와 무관한 다른 세포내 흡수경로를 이용하는 것을 시사한다. 대사억제제의 경우, DA3 회합체의 트랜스팩션 효율이 약 73%로 감소되었으나, PEI 25 kDa의 경우는 약 99.6%로 감소되었다. 따라서, 본 발명에 따른 회합체는 거대분자, 예컨대 플라스미드 DNA와 같은 분자를, 엔도시토시스 경로나 다른 에너지 소비성 세포내 흡수 경로의 도움 없이 세포내로 전달할 수 있음을 확인할 수 있다. As shown in FIG. 5, each inhibitor and combination of inhibitors did not appear to have a significant effect on the transfection of the DA3 association. This suggests that the DA3 assembly utilizes other intracellular uptake pathways independent of endocytosis. In the case of metabolic inhibitors, the transfection efficiency of the DA3 association was reduced to about 73%, whereas for PEI 25 kDa it was reduced to about 99.6%. Thus, it can be seen that the assembly according to the present invention can deliver macromolecules, such as molecules such as plasmid DNA, intracellularly without the help of endocytosis pathways or other energy consuming intracellular uptake pathways.

3) 세포독성 실험3) Cytotoxicity Test

10% FBS를 포함하는 DMEM 0.2 ml의 성장 배지(growth medium)가 있는 96 well plate에 세포를 5000 cells/well의 밀도로 접종하였다. 세포는 37℃에서 24시간 동안 성장시켰고, 성장배지를 제거하고 적당량의 폴리머를 포함하는 성장배지를 공급하였다. 추가로 세포를 48시간 배양한 후, 세포의 대사활성(metabolic activity)을 측정하였다. 50 μg MTT를 포함하는 100 μL의 성장배지를 각 well에 첨가한 후 4시간동안 세포를 배양하였다. Lysis 버퍼(10 % (w/v) SDS, 45 % DMF, pH 4.7)를 첨가한 후 24시간 동안 배양하였다. Microplate reader(EL808, Bio-Tek Instrument, Winooski, VT)로 590 nm에서의 흡수를 측정하였고, 생존률을 mock-treated cells(100 % 생존)과 비교하여 계산하였다. 그 결과를 도 6에 나타내었다. Cells were seeded at a density of 5000 cells / well in 96 well plates with 0.2 ml of DMEM growth medium containing 10% FBS. Cells were grown for 24 hours at 37 ° C., the growth medium was removed and a growth medium containing an appropriate amount of polymer was fed. After incubating the cells for 48 hours, the metabolic activity of the cells was measured. 100 μL of growth medium containing 50 μg MTT was added to each well and the cells were incubated for 4 hours. Lysis buffer (10% (w / v) SDS, 45% DMF, pH 4.7) was added and incubated for 24 hours. Absorption at 590 nm was measured with a microplate reader (EL808, Bio-Tek Instrument, Winooski, VT), and survival was calculated by comparison with mock-treated cells (100% survival). The results are shown in FIG.

도 6에 나타난 바와 같이, 회합체가 PEI25KDa 보다 낮은 독성을 나타내며, PEI1.8보다는 높은 독성을 나타내지만 이는 회합체의 특성 중 하나인 세포막활성에 기인한 것으로 판단된다. As shown in FIG. 6, the association shows lower toxicity than PEI25KDa and higher toxicity than PEI1.8, but this may be due to cell membrane activity, which is one of the characteristics of the association.

4) 중간엽 줄기 세포의 분리, 배양 및 transfection4) Isolation, Culture and Transfection of Mesenchymal Stem Cells

골수 유래 중간엽 줄기세포(bone marrow-derived mesenchimal stem cells (MSCs))를 4주 Sprague-Dawley rats의 대퇴부 및 경골로부터 종래문헌을 참조하여 magnetic bead-based cell isolation 방법으로 분리하였다(Song, H.; Chang, W.; Lim, S.; Seo, H.-S.; Shim, C. Y.; Park, S.; Yoo, K.-J.; Kim, B.-S.; Min, B.-H.; Lee, H.; Jang, Y.; Chung, N.; Hwang, K.-C. Stem Cells 2007, 25, 1431-1438). 분리된 MSCs를 10% FBS를 포함하는 저농도 글루코즈 DMEM에서 37℃ 및 5% CO2 조건을 유지하면서 배양하였다. CD14, CD34, CD71, CD90, CD105, 및 세포간 접착분자(intracellular adhesion molecules; ICAM-1)를 포함하는 일련의 항체 표적 세포내 마커(antibodies targeting cellular markers)를 이용하여 MSCs를 flow cytometry로 특정하였다. Transfection 실험을 위하여, 세포를 트립신화하고, 6-well plate에 접종한 후(1×105 cells/well), 37℃ 및 5% CO2 조건을 유지하면서 배양하였다. 24시간 배양후에, DA3 회합체, 가지쇄 PEI 25 kDa (b-PEI25K), 선형 PEI 25 kDa (l-PEI25K) 및 Lipofectamine-Plus™(Invitrogen, Carlsbad, CA)을 이용하여 transfection 시험을, 상기 1)의 방법과 동일하게 수행하였다. 그 결과를 도 7에 나타내었다. Bone marrow-derived mesenchimal stem cells (MSCs) were isolated from the femoral and tibia of 4 week Sprague-Dawley rats by magnetic bead-based cell isolation (Song, H.). ; Chang, W .; Lim, S .; Seo, H.-S .; Shim, CY; Park, S .; Yoo, K.-J .; Kim, B.-S .; Min, B.-H Lee, H .; Jang, Y .; Chung, N .; Hwang, K.-C. Stem Cells 2007, 25, 1431-1438). Isolated MSCs were incubated in low glucose DMEM with 10% FBS, maintaining 37 ° C. and 5% CO 2 conditions. MSCs were characterized by flow cytometry using a series of antibody targeting cellular markers including CD14, CD34, CD71, CD90, CD105, and intracellular adhesion molecules (ICAM-1). . For transfection experiments, cells were trypsinized, inoculated into 6-well plates (1 × 10 5 cells / well), and incubated at 37 ° C. and 5% CO 2 conditions. After 24 hours of incubation, transfection test using DA3 association, branched PEI 25 kDa (b-PEI25K), linear PEI 25 kDa (l-PEI25K) and Lipofectamine-Plus ™ (Invitrogen, Carlsbad, Calif.), 1 Was carried out in the same manner as). The results are shown in FIG.

도 7에 도시된 바와 같이, DA3 회합체는 가지쇄 PEI 25 kDa (b-PEI25K), 선형 PEI 25 kDa (l-PEI25K) 및 Lipofectamine-Plus™ 각각에 대해 10배, 6배, 15배의 높은 트랜스팩션 효율을 나타내었다. 따라서 본 발명에 따른 회합체는 융합세포 뿐만 아니라 primary cells에서도 세포내 플라스미드 DNA의 전달을 매개할 수 있음을 나타낸다. As shown in FIG. 7, the DA3 association was 10, 6 and 15 times higher for branched PEI 25 kDa (b-PEI25K), linear PEI 25 kDa (l-PEI25K) and Lipofectamine-Plus ™, respectively. Transfection efficiency is shown. Therefore, the association according to the present invention indicates that it can mediate the delivery of intracellular plasmid DNA in primary cells as well as fusion cells.

도 1은 본 발명의 DA-PEI 회합체의 합성반응을 도시한 것이다. Figure 1 shows the synthesis of the DA-PEI assembly of the present invention.

도 2는 본 발명의 DA1 및 DA3의 치환된 정도를 확인하기 위한 1H-NMR결과를 나타낸 것이다. Figure 2 shows the 1H-NMR results to confirm the degree of substitution of DA1 and DA3 of the present invention.

도 3은 본 발명에 따른 복합체의 이동 지연을 ethidium bromide staining으로 영상화한 것이다. Figure 3 is an image of the migration delay of the complex according to the present invention by ethidium bromide staining.

도 4는 본 발명에 따른 회합체의 Transfection 및 reporter gene assay 결과를 나타낸 것이다. Figure 4 shows the results of the transfection and reporter gene assay of the assembly according to the present invention.

도 5는 본 발명에 따른 회합체의 억제제의 첨가에 따른 Transfection 및 reporter gene assay 결과를 나타낸 것이다. Figure 5 shows the results of the transfection and reporter gene assay according to the addition of the inhibitor of the association according to the present invention.

도 6은 본 발명에 따른 회합체의 세포독성에 대한 결과를 나타낸 것이다. Figure 6 shows the results for the cytotoxicity of the assembly according to the present invention.

도 7은 중간엽 줄기 세포에 대한 본 발명의 transfection 결과를 나타낸 것이다. Figure 7 shows the transfection results of the present invention for mesenchymal stem cells.

Claims (9)

폴리에틸렌이민(PEI) 및 담즙산으로 구성된 회합체에 있어서, 상기 담즙산의 카르복시기와 폴리에틸렌이민의 아민기의 반응에 의하여 결합된 회합체.An association composed of polyethyleneimine (PEI) and bile acid, wherein the association is performed by reaction of a carboxyl group of the bile acid with an amine group of polyethyleneimine. 제1항에 있어서, 상기 담즙산은 콜린산(cholic acid), 5β-콜란산(5β-cholanic acid), 체노데옥시콜린산(chenodeoxycholic acid), 글리코콜린산(glycocholic acid), 타우로콜린산(taurocholic acid), 데옥시콜린산(deoxycholic acid), 리쏘콜린산(lithocholic acid) 및 7-옥소-리소콜린산(7-oxo-lithocholic acid)으로 구성되는 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 회합체.According to claim 1, The bile acid is cholic acid (cholic acid), 5β-cholanic acid (5β-cholanic acid), chenodeoxycholic acid (chenodeoxycholic acid), glycocholic acid (glycocholic acid), taurocholic acid ( taurocholic acid, deoxycholic acid, lithocholic acid and 7-oxo-lithocholic acid, characterized in that any one selected from the group consisting of Association. 제1항에 있어서, 상기 폴리에틸렌이민의 분자량은 10 내지 100kDa인 것을 특징으로 하는 회합체.The assembly of claim 1, wherein the polyethyleneimine has a molecular weight of 10 to 100 kDa. 제3항에 있어서, 상기 폴리에틸렌이민의 분자량은 1.8 kDa인 것을 특징으로 하는 회합체.4. The association according to claim 3, wherein the polyethyleneimine has a molecular weight of 1.8 kDa. 제1항에 있어서, 상기 담즙산과 상기 폴리에틸렌이민의 몰 비는 1 내지 50%인 것을 특징으로 하는 회합체.The assembly of claim 1, wherein the molar ratio of the bile acid to the polyethyleneimine is 1-50%. 제1항 내지 제5항 중 어느 한 한의 회합체에 거대분자 또는 소수성 약물이 봉입되어 있는 것을 특징으로 하는 복합체.The complex according to any one of claims 1 to 5, wherein a macromolecule or a hydrophobic drug is encapsulated. 제6항에 있어서, 상기 거대분자는 플라스미드 DNA, siRNA 및 안티센스올리고뉴클레오티드로 구성되는 군으로부터 선택되는 어느 하나 인것을 특징으로 하는 복합체.The complex of claim 6, wherein the macromolecule is any one selected from the group consisting of plasmid DNA, siRNA, and antisense oligonucleotides. 제6항에 있어서, 상기 소수성 약물로는 독소루비신(doxorubicin), 아드리아마이신(adriamycin), 시스플라틴(cisplatin), 파클리탁셀(paclitaxel), 탁솔(taxol), 도세탁셀(docetaxel), 다우노마이신(daunomycin), 조효소 Q10(coenzyme Q10), 비타민 E 및 그의 유도체, 비타민 A 및 그의 유도체로 구성되는 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 복합체.According to claim 6, wherein the hydrophobic drug (doxorubicin), adriamycin (adriamycin), cisplatin (cisplatin), paclitaxel (taxita), taxol (docetaxel), daunoomycin (daunomycin), coenzyme Q10 (coenzyme Q10), vitamin E and its derivatives, vitamin A and any one selected from the group consisting of derivatives thereof. 담즙산을 디사이클로헥실 카르보디이미드(dicyclohexyl carbodiimide; DCC) 및 N-하이드록시 석신이미드(N-hydroxyl succinimide; NHS)로 활성화시키는 단계; 및Activating bile acids with dicyclohexyl carbodiimide (DCC) and N-hydroxys succinimide (NHS); And 상기 활성된 담즙산에 폴리에틸렌이민을 첨가하는 단계를 포함하는 폴리에틸렌이민(PEI) 및 담즙산으로 구성된 회합체의 제조방법.Polyethyleneimine (PEI) comprising the step of adding polyethyleneimine to the activated bile acid and a method for producing an assembly consisting of bile acid.
KR1020090118416A 2009-12-02 2009-12-02 Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity KR101128108B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020090118416A KR101128108B1 (en) 2009-12-02 2009-12-02 Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020090118416A KR101128108B1 (en) 2009-12-02 2009-12-02 Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity

Publications (2)

Publication Number Publication Date
KR20110061881A true KR20110061881A (en) 2011-06-10
KR101128108B1 KR101128108B1 (en) 2012-03-22

Family

ID=44396161

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020090118416A KR101128108B1 (en) 2009-12-02 2009-12-02 Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity

Country Status (1)

Country Link
KR (1) KR101128108B1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10350169B2 (en) 2014-10-31 2019-07-16 University Of Utah Research Foundation Compositions and methods for bile acid particles
KR102299170B1 (en) * 2021-01-13 2021-09-07 영남대학교 산학협력단 Polyethyleneimine-cholic acid ionic bonding compound with gene transfer ability and use thereof
US11266747B2 (en) 2016-04-26 2022-03-08 Kb Biomed Inc. Orally administered nanoparticles for gene delivery and pharmaceutical composition containing same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100904207B1 (en) 2007-06-01 2009-06-25 (주) 태웅메디칼 Coating agent for drug releasing stent, manufacturing method thereof and drug releasing stent coated with the coating agent
KR100964372B1 (en) * 2007-12-04 2010-06-17 가톨릭대학교 산학협력단 Complex for treating a cancer, a process for the preparation thereof, and an injection comprising the same

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10350169B2 (en) 2014-10-31 2019-07-16 University Of Utah Research Foundation Compositions and methods for bile acid particles
US11266747B2 (en) 2016-04-26 2022-03-08 Kb Biomed Inc. Orally administered nanoparticles for gene delivery and pharmaceutical composition containing same
KR102299170B1 (en) * 2021-01-13 2021-09-07 영남대학교 산학협력단 Polyethyleneimine-cholic acid ionic bonding compound with gene transfer ability and use thereof
WO2022154239A1 (en) * 2021-01-13 2022-07-21 영남대학교 산학협력단 Ionically bonded compound of polyethylenimine-cholic acid with gene transfer activity and use thereof

Also Published As

Publication number Publication date
KR101128108B1 (en) 2012-03-22

Similar Documents

Publication Publication Date Title
Gao et al. Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency
Jiang et al. Overcoming drug-resistant lung cancer by paclitaxel loaded dual-functional liposomes with mitochondria targeting and pH-response
Kibria et al. Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery
Pu et al. Dual-targeting liposomes with active recognition of GLUT5 and αvβ3 for triple-negative breast cancer
Guo et al. Cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector
Liu et al. Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly (lactic acid)–poly (ethylene glycol) nanoparticles through caveolae-mediated endocytosis
Xu et al. Efficient and targeted drug/siRNA co-delivery mediated by reversibly crosslinked polymersomes toward anti-inflammatory treatment of ulcerative colitis (UC)
JP2010526091A (en) Modification of biological target groups for the treatment of cancer
JP2010526091A5 (en)
Li et al. GSH/pH dual-responsive biodegradable camptothecin polymeric prodrugs combined with doxorubicin for synergistic anticancer efficiency
dos Santos Rodrigues et al. In vitro and in vivo characterization of CPP and transferrin modified liposomes encapsulating pDNA
Gao et al. A progressively targeted gene delivery system with a pH triggered surface charge-switching ability to drive angiogenesis in vivo
Lee et al. Gene delivery of PAMAM dendrimer conjugated with the nuclear localization signal peptide originated from fibroblast growth factor 3
Gaspar et al. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery
He et al. Gene delivery with active targeting to ovarian cancer cells mediated by folate receptor α
WO2017002979A1 (en) Drug delivery carrier, and composition containing same
Mallick et al. Polyamidoamine (PAMAM) dendrimers modified with short oligopeptides for early endosomal escape and enhanced gene delivery
Abraham et al. Nanoliposomes for safe and efficient therapeutic mRNA delivery: a step toward nanotheranostics in inflammatory and cardiovascular diseases as well as cancer
Hussein et al. Peptide-based targeted polymeric nanoparticles for siRNA delivery
Yang et al. pH and redox dual-responsive multifunctional gene delivery with enhanced capability of transporting DNA into the nucleus
Choi et al. Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency
Azimifar et al. Evaluation of the efficiency of modified PAMAM dendrimer with low molecular weight protamine peptide to deliver IL‐12 plasmid into stem cells as cancer therapy vehicles
KR20140037449A (en) Hyaluronic acid-nucleic acid conjugate and composition for nucleic acid delivery containing the same
Jain et al. Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro
Yeh et al. Stimuli-responsive HA-PEI nanoparticles encapsulating endostatin plasmid for stem cell gene therapy

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20150115

Year of fee payment: 4

FPAY Annual fee payment

Payment date: 20160113

Year of fee payment: 5

FPAY Annual fee payment

Payment date: 20170308

Year of fee payment: 6

FPAY Annual fee payment

Payment date: 20180528

Year of fee payment: 7