고분자(PLL, PEI, Chitosan) 나노복합체를 이용한 유전자 전달체

소개

유전자 전달용 고분자 (Polyplex)의 종류에는 Poly-L-lysine (PLL), Polyethylenimine (PEI), Chitosan 이 있다. 하지만 유전자의 전달 효율이 낮다는 문제가 있어 비 바이러스계 전달체에 의한 유전자 전달의 여러 단계중 각기 다른 단계에서 작용하는 고분자 물질을 조합하는 것이 현재 연구동향이다. DNA / PEI / Chitosan 복합체는 100%의 혈청 조건에서 chitosan이 첨가되면서 단독 PEI 전달체에 비해 유전자 전달 효율이 약 10배정도 증가 되며, DNA / protamine sulfate / Chitosan / KALA 복합체는 45 kDa의 chitosan 조건에서 최적의 중량비는 DNA/chitosan=1:4이고, PS와 KALA peptide을 병행 하였을 때 약 200배 이상의 유전자 전달 효율이 증가된다. AWBP-PEG-g-PLL 복합체는 PLL/pDNA와 PEG-g-PLL/pDNA와 같은 기존의 시스템이 가진 전이효율보다 150~180배정도 더 높게 나타난다.

목차

1. 서 론 ······································································································· 7

2. History ···································································································· 9

3. 바이러스성 벡터와 비바이러스성 벡터 ····························································· 10

4. 유전자 전달용 고분자 (Polymer/DNA Complex : Polyplex) ································· 11
4.1. Poly-L-lysine (PLL) ·············································································· 12
4.2. Polyethylenimine (PEI) ··········································································· 13
4.3. Chitosan ·····························································································15

5. DNA-polymer 복합체 제조 ·········································································· 16
5.1. DNA / AWBP-PEG-g-PLL 복합체 ···························································· 16
5.1.1. AWBP-PEG-g-PLL 복합체 제조 ························································ 16
5.1.2. 유전자 발현에 대한 분석 ·································································· 17
5.2. DNA / PEI / Chitosan 복합체 ·································································· 20
5.2.1 제조 ··························································································· 20
5.2.2 PEI와 chitosan의 비율에 따른 DNA-polymer 복합체의 유전자 전달 효율 ········ 21
5.2.3. PEI 매개 복합체에 PLL 또는 chitosan의 첨가에 따른 유전자 전달 효율 ········· 22
5.2.4. DNA-polymer 복합체의 세포독성 측정·················································· 23
5.3. DNA / protamine sulfate / Chitosan / KALA 복합체 ······································ 24
5.3.1. Meterial ······················································································ 24
5.3.1.1. Protamine sulfate (PS) ······························································ 24
5.3.1.2. KALA ·················································································· 25
5.3.2. 제조 ·························································································· 25
5.3.3. Chitosan 분자량과 중량비율에 따른 transfection 효율 ······························· 26
5.3.4. PS와 chitosan의 첨가 비율에 따른 transfection 효율 ································ 26
5.3.5. DNA/PS/chitosan 복합체에 KALA peptide의 코팅에 따른 유전자 전달 효율 ··· 28

6. 결론 ······································································································ 29

7. 참고자료 ································································································· 30

본문내용

DNA는 세포질에서 불안정하기 때문에 빠른 시간에 핵으로 이동되어야 하는데 유전자 전달용 고분자는 핵으로의 유전자 전달을 도우며, 핵산 분해효소로부터 방어한다는 이점을 바탕으로 많은 연구가 진행되고 있다. 고분자성 물질로는 Polyethylenimine (PEI), Poly-L-lysine (PLL), Chitosan등이 있는데 각각의 하나의 물질로만 DNA와 축합시켜 세포내이입을 할 경우 전달효율성 및 독성 등의 문제가 생기기 때문에 여러 가지 고분자를 복합하여 사용되고 있다.
4.1. Poly-L-lysine (PLL)
양이온성 고분자인 PLL은 최초로 사용된 유전자 전달용 고분자 중의 하나이며 다양한 크기의 분자량으로 자유자재로 합성할 수 있기 때문에 많은 연구가 진행되고 있다. Figure 2.은 poly-L-lysine (PLL)의 화학구조이다. PLL은 생분해성인 펩타이드 결합으로 연결되어 있기 때문에 in vivo에 활발히 응용되고 있으며 유전자와 나노복합체를 형성해서 세포내로 전달될 수 있다. 포리라이신에 존재하는 아민그룹은 중성 pH에서는 양전하 ( )를 띠게 된다. 중성 pH에서 전달체가 띠는 양전하는 유전자의 음전하와 이온결합이 가능하게 되며 이러한 결합력을 이용하여 유전자를 작은 크기로 압축시키게 된다. 그러나 어느 정도의 독성도 나타내고 있으며 PLL은 단독으로는 높은 전달효율을 나타내는 데는 한계가 있다. 이를 극복하기 위해 엔도좀 파괴 물질이나 엔도좀 융합펩타이드 등을 부착시킴으로써 높은 전달효율을 보여주고 있다. 또한 PLL에 다당류(polysaccharide)를 결합시킨 공중합체를 이용하여 지름이 약 100nm이하인 nanoparticle 을 만들어 이용하기도 하였다. 그리고 PLL 의 in vivo circulation을 더 길게 하기 위한 노력으로 polyethylene glycol(PEG)를 결합시킨 PEG-g-PLL도 합성되어 유전자 전달 효율도 입증된 바가 있다.[8]

참고자료

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· 13. I. K. Park, T. H. Kim, S. I. Kim, Y. H. Park, W. J. Kim, T. Akaike, and C. S. Cho, Int. J. Pharm., 257, 103 (2003).