생체 적합성을 지니는 글리콜 키토산을 기반으로 하는 나노입자 제조를 통한 유전자 전달체 연구

이영화; 최준식; Young Hwa Lee; Joon Sig Choi

Polymer(Korea), Vol.42, No.5, 849-856, September, 2018

DOI10.7317/pk.2018.42.5.849 Export Citation

생체 적합성을 지니는 글리콜 키토산을 기반으로 하는 나노입자 제조를 통한 유전자 전달체 연구

Biocompatible Glycol Chitosan-based Nanoparticles for Gene Delivery System

이영화, 최준식^†

충남대학교 자연과학대학 생화학과

Young Hwa Lee, and Joon Sig Choi^†

Department of Biochemistry, Chungnam National University, Gung-dong 220, Yuseong-gu, Daejeon 34134, Korea

E-mail:

초록

최근 많은 연구를 통해 유전자 전달체로써 생체 적합하며 생분해성이 뛰어난 키토산에 대한 연구가 활발히 진행되고 있다. 본 연구에서는 키토산의 유도체인 글리콜 키토산을 사용하여 메틸 아크릴레이트와 폴리에틸렌이민을 도입하여 유전자 전달체를 개발 및 연구하였다. 최종 합성물인 GMP 1300과 GMP 2000은 핵자기 공명 분광기를 이용해 합성 정도를 평가하였고, 플라스미드 DNA와의 결합력 확인을 위해 전기영동을 수행하였다. 그리고 나노 입자 분석기를 이용하여 플라스미드 DNA와 결합시킨 GMP 1300과 GMP 2000의 입자 크기와 표면 전하를 측정하였다. In vitro 실험을 위하여, HeLa와 HEK 293 세포주를 이용하여 세포 독성 평가를 진행하였고, 세포 유입은 공초점 현미경을 통해 확인하였다. 또한 유전자 발현율을 평가하기 위해 녹색 형광 단백질을 발현하여 이미지로 확인 하였고, 루시페라아제와 단백질 정량을 통해 유전자 발현 효율을 측정하였다. 그 결과, 본 연구에서 제조된 나노 입자들은 독성이 현저히 낮으며, 유전자와의 결합력이 강하고, 세포 내 유입에 용이하다는 것을 통해 생체적합한 유전자 전달체로써 응용될 수 있을 것으로 기대된다.

Since chitosan is biocompatible and biodegradable, it has been studied as a good material as a gene delivery carrier. In this study, we have developed and studied a gene delivery carrier by introducing methyl acrylate and polyethylenimine using glycol chitosan, a derivative of chitosan. GMP1300 and GMP2000 were identified by 1H NMR and electrophoresis was performed to confirm the binding ability with pDNA. The particle size and surface charge of GMP1300 and GMP2000 with pDNA were measured using a zetasizer. Cytotoxicity was assessed using HeLa and HEK293 cell lines, and cellular uptake was confirmed by confocal microscopy. In addition, the gene expression rate in HeLa cell line was confirmed by confocal image through green fluorescent protein (GFP), and transfection efficiency was measured by protein quantification. As a result, the prepared nanoparticles were expected to be used as biocompatible gene delivery carriers because of their low toxicity and strong binding force with genes.

Keywords:gene delivery;cationic polymer;glycol chitosan;polyethylenimine;transfection

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Lee YH, Yim KH, Heo J, Choi JS, Polym. Korea, 38(1), 43 (2014)
Jiang MY, Wang KM, Kennedy JF, Nie J, Yu QA, Ma GP, Int. J. Biol. Macromol., 47, 696 (2010)
Lee YH, Park HI, Choi JS, Carbohydr. Polym., 137, 669 (2016)
Yu JM, Xie X, Zheng MR, Yu L, Zhang L, Zhao JG, Jiang DZ, Che XX, Int. J. Nanomed., 7, 5079 (2012)
Huh MS, Lee SY, Park S, Lee S, Chung H, Lee S, Choi Y, et al., J. Control. Release, 144, 134 (2010)
Honore I, Grosse S, Frison N, Favatier F, Monsigny M, Fajac I, J. Control. Release, 107, 537 (2005)
Hufnagel H, Hakim P, Lima A, Hollfelder F, Mol. Ther., 17, 1411 (2009)

[1] Kataoka K, Harashima H, Adv. Drug Deliv. Rev., 52, 151 (2001)

[2] Cristiano RJ, Anticancer Res., 18, 3241 (1998)

[3] Azzoni AR, Wu ML, Freitas SS, Monteiro GA, Prazeres DMF, Human Gene Therpy, 18, 1053 (2007)

[4] Benns JM, Choi JS, Mahato RI, Park JS, Kim SW, Bioconjug. Chem., 11, 637 (2000)

[5] Lim YB, Kim SM, Suh H, Park JS, Bioconjug. Chem., 13, 952 (2002)

[6] Ye ZY, Kumar N, Narang AS, Thoma L, Miller DD, Mahato RI, Molecular Therapy, 7, S216 (2003)

[7] Patel MP, Patel RR, Patel JK, J. Pharm. Pharm. Sci.

[8] Moghadas B, Dashtimoghadam E, Mirzadeh H, Seidi F, Hasani-Sadrabadi MM, RSC Adv, 6, 7701 (2016)

[9] Khor E, Lim LY, Biomaterials, 24, 2339 (2003)

[10] Dai JA, Zou SY, Pei YY, Cheng D, Ai H, Shuai XT, Biomaterials, 32, 1694 (2011)

[11] Chen XA, Zhang LJ, He ZJ, Wang WW, Xu B, Zhong Q, Shuai XT, Yang LQ, Deng YB, Int. J. Nanomed., 6, 843 (2011)

[12] Son S, Kim WJ, Biomaterials, 31, 133 (2010)

[13] Lee YH, Yim KH, Heo J, Choi JS, Polym. Korea, 38(1), 43 (2014)

[14] Jiang MY, Wang KM, Kennedy JF, Nie J, Yu QA, Ma GP, Int. J. Biol. Macromol., 47, 696 (2010)

[15] Lee YH, Park HI, Choi JS, Carbohydr. Polym., 137, 669 (2016)

[16] Yu JM, Xie X, Zheng MR, Yu L, Zhang L, Zhao JG, Jiang DZ, Che XX, Int. J. Nanomed., 7, 5079 (2012)

[17] Huh MS, Lee SY, Park S, Lee S, Chung H, Lee S, Choi Y, et al., J. Control. Release, 144, 134 (2010)

[18] Honore I, Grosse S, Frison N, Favatier F, Monsigny M, Fajac I, J. Control. Release, 107, 537 (2005)

[19] Hufnagel H, Hakim P, Lima A, Hollfelder F, Mol. Ther., 17, 1411 (2009)