시스타민 코어 폴리아미도아민 덴드리머에 펩티드와 감마 아미노부티르산의 도입을 통한 새로운 유전자 전달체 연구

박혜원; 유광식; 송수정; 최준식; Hye Won Park; Gwang Sig Yu; Su Jeong Song; Joon Sig Choi

Polymer(Korea), Vol.39, No.5, 727-732, September, 2015

DOI10.7317/pk.2015.39.5.727 Export Citation

시스타민 코어 폴리아미도아민 덴드리머에 펩티드와 감마 아미노부티르산의 도입을 통한 새로운 유전자 전달체 연구

Conjugation of Peptide to Cystamine Core Polyamidoamine with γ-Aminobutyric Acid for Gene Delivery

박혜원, 유광식, 송수정, 최준식^†

충남대학교 자연과학대학 생화학과

Hye Won Park, Gwang Sig Yu, Su Jeong Song, and Joon Sig Choi^†

Department of Biochemistry, Chungnam National University, Gung-dong 220, Yuseong-gu, Daejeon 305-764, Korea

E-mail:

초록

유전자 전달체 가운데 비바이러스성 전달체인 PAMAM 덴드리머의 일종인 cPAMAM G4를 사용하여 분자의 표면에 GABA와 히스티딘, 아르기닌을 도입해 새로운 유전자 전달체를 합성하였다. cPAMAM G4-GABA-HR 최종합성물은 핵자기 공명 분광기를 이용해 합성률을 확인하였다. 전기영동을 이용하여 폴리머와 pDNA간의 결합력을 확인하였고, 폴리머와 DNA의 복합체에 환원제를 처리하여 폴리머의 pDNA 방출 능력을 확인하였다. 나노 입자 분석기와 원자력 현미경을 이용하여 pDNA와 폴리머 복합체의 크기와 모양을 분석하였다. HeLa 세포주를 이용한 WST-1 assay를 통해 폴리머의 세포 독성을 확인하였고, 동일한 세포주를 이용한 luciferase assay를 통해 유전자 전달 효율성을 확인하였다.

Polyamidoamine (PAMAM) dendrimer are well known non-viral vectors for gene delivery application. Especially, PAMAM conjugated with amino acids have enhanced cell uptake capability and low cytotoxicity compared to other non-viral, vectors. Moreover cystamine core PAMAM has an ability to degrade inside low pH of endosome leading to ease release of biomolecules. In present work, we designed γ-amino butyric acid (GABA), histidine, arginine modified cystamine core PAMAM. GABA has four carbon chains that plays a role as flexible linkers, hence easy interaction with cell membrane. cPAMAM-GABA-HR synthesis was confirmed by 1H NMR. We performed gel retardation assay, DNA release phenomenon of cystamine core PAMAM using DTT. We measured size of polyplex by DLS and AFM. Cytotoxicity was examined by WST-1 assay and transfection efficiency was confirmed using reporter plasmid in HeLa cells.

Keywords:cationic polymer;PAMAM dendrimer;transfection;amino acid

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[Referenced By]

[1] Friedmann T, Trends Biotechnol., 11, 156 (1993)

[2] Rao RC, Zacks DN, Dev. Ophthalmol., 53, 167 (2014)

[3] Zhang T, Curr. Drug Deliv., 11, 233 (2014)

[4] Cristiano RJ, Front. Biosci., 3, D1161 (1998)

[5] Nayerossadat N, Maedeh T, Ali P, Adv. Biomed. Res., 1, 27 (2012)

[6] Lentz TB, Gray SJ, Samulski RJ, Neurobiol. Dis., 48, 179 (2012)

[7] Hewinson J, Paton JFR, Kasparov S, Methods Mol. Biol., 998, 65 (2013)

[8] Dutta T, Jain NK, McMillan NAJ, Parekh HS, Nanomedicine, 6, 25 (2010)

[9] Almofti MR, Harashima H, Shinohara Y, Almofti A, Baba Y, Kiwada H, Arch. Biochem. Biophys., 410, 246 (2003)

[10] Hattori Y, Maitani Y, Curr. Drug Deliv., 2, 243 (2005)

[11] Menjoge AR, Kannan RM, Tomalia DA, Drug Discov. Today, 15, 171 (2010)

[12] Malik N, Wiwattanapatapee R, Klopsch R, Lorenz K, Frey H, Weener JW, Meijer EW, Paulus W, Duncan R, J. Contr. Rel., 65, 133 (2000)

[13] Patil ML, Zhang M, Betigeri S, Taratula O, He H, Minko T, Bioconjug. Chem., 19, 1396 (2008)

[14] Neumann H, Smith RA, Arch. Biochem. Biophys., 122, 354 (1967)

[15] Yu GS, Yu HN, Choe YH, Son SJ, Ha TH, Choi JS, Bull. Korean Chem. Soc., 32, 651 (2011)

[16] Varkouhi AK, Scholte M, Storm G, Haisma HJ, J. Contr. Rel., 151, 220 (2011)

[17] Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC, Jones AT, Sugiura Y, Futaki S, Mol. Ther., 10, 1011 (2004)

[18] Maiolo JR, Ferrer M, Ottinger EA, Biochim. Biophys. Acta, 1712, 161 (2005)

[19] Yu GS, Bae YM, Kim JY, Han J, Ko KS, Choi JS, Macromol. Res., 20(11), 1156 (2012)