期刊
JOURNAL OF GENE MEDICINE
卷 5, 期 3, 页码 232-245出版社
WILEY
DOI: 10.1002/jgm.331
关键词
cancer; DNA; gene therapy; lipid; nitroreductase; poly(L-lysine)
Background Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys(10)-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techiques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presences of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. Results A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 +/- 6.7%) and PC-3 (66.1 +/- 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 result in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery on nucleic acids and potentiate therapeutic activity. Copyright (C) 2002 John Wiley & Sons, Ltd.
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