caa a22 4500 445313250 CHVBK 20180317142637.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s11095-010-0335-z doi (NATIONALLICENCE)springer-10.1007/s11095-010-0335-z Efficient Intracellular Gene Delivery Using the Formulation Composed of Poly (L-glutamic Acid) Grafted Polyethylenimine and Histone [Elektronische Daten] [Jingjing Deng, Yuting Wen, Cuifeng Wang, Shirong Pan, Huaiyu Gu, Xin Zeng, Lina Han, Yuefang Zhao, Min Feng, Chuanbin Wu] ABSTRACT: Purpose: Inefficient endosomal escape and poor nuclear import are thought to contribute to low gene transfer efficiency of polycations. To overcome these drawbacks, we prepared multiple gene delivery formulations including low cytotoxic polycation, histone containing NLSs and chloroquine as the endosomolytic agent. Methods: Comb-shaped poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) copolymer was synthesized by aminolysis of poly-γ-benzyl-L-glutamate using low-molecular-weight polyethylenimine (800Da). The formation of DNA/histone/PLGE terplex was observed by atomic force microscope and gel retardation assay. The particle size and zeta potential of DNA complexes with varying content of histone were also measured to confirm the terplex formation. Cytotoxicity of vectors was assayed by MTT. Multiple gene delivery formulations were optimized to their best transfection efficiency that was monitored by fluorescence microscope and flow cytometry. In vivo gene delivery of the optimal formulation was evaluated by the GFP-expression levels in drosophila melanogaster. Results: The DNA/histone/PLGE terplex was successfully formed. The PLGE and histone together condensed DNA into small, discrete particles (less than 200nm in diameter) in isotonic solution. Cytotoxicity of PLGE and histone were much lower than that of PEI 25K. Either histone or chloroquine contributed to enhancing the levels of transfection activity of PLGE polymer. However, chloroquine and histone did not show a synergistic effect on the improvement of transfection efficiency. The optimal formulation was the DNA/histone/PLGE terplex at the N/P ratio of 15 and histone/ DNA weight ratio of 0.8. Compared with Lipofectamine 2000 and PEI 25K, the optimal formulation showed significantly increased levels of GFP-expression both in vitro and in vivo. Conclusion: This formulation provided a versatile approach for preparing high efficiency of the polycation-based gene vectors. It also reinforced the finding of earlier studies that nuclear import and endosomal escape were rate-limiting steps for nonviral gene delivery. Springer Science+Business Media, LLC, 2010 chloroquine nationallicence gene delivery formulation nationallicence histone nationallicence poly (L-glutamic acid) nationallicence polyethylenimine nationallicence transfection efficiency nationallicence 2-HP : 2-Hydroxypyridine nationallicence DMSO : Dimethyl sulfoxide nationallicence FBS : fetal bovine serum nationallicence GFP : green florescent protein nationallicence MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide nationallicence NLS : nuclear localization signal nationallicence PBLG : poly-γ-benzyl-L-glutamate nationallicence pDNA : plasmid DNA nationallicence PEI 25K : polyethylenimine with molecular weight of 25KDa nationallicence PLGE : poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine nationallicence Deng Jingjing Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Wen Yuting Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Wang Cuifeng Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Pan Shirong Cardiovascular Laboratory, The First Affiliated Hospital, Sun Yat-sen University, 80 Zhongshan Road II, 510080, Guangzhou, People's Republic of China aut Gu Huaiyu Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Zeng Xin Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Han Lina Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Zhao Yuefang Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Feng Min Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Wu Chuanbin Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/4(2011-04-01), 812-826 0724-8741 28:4<812 2011 28 11095 https://doi.org/10.1007/s11095-010-0335-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-010-0335-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Deng Jingjing Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Wen Yuting Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Wang Cuifeng Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Pan Shirong Cardiovascular Laboratory, The First Affiliated Hospital, Sun Yat-sen University, 80 Zhongshan Road II, 510080, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Gu Huaiyu Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Zeng Xin Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Han Lina Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Zhao Yuefang Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Feng Min Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Wu Chuanbin Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, 510006, Guangzhou, People's Republic of China aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/4(2011-04-01), 812-826 0724-8741 28:4<812 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer