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KMID : 0369820130430060499
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Jorunal of Korean Pharmaceutical Sciences
2013 Volume.43 No. 6 p.499 ~ p.505
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Preliminary study to determine the optimal conditions for the simultaneous complexation of siRNA and plasmid DNA
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Davaa Enkhzaya
Ahn Ick-Hyun
Kang Bong-Seok
Lee Sang-Eun
Myung Chang-Seon
Park Jeong-Sook
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Abstract
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We investigated the complexation ratio of siRNA and plasmid DNA combined with Lipofectamine for the effective delivery of genetic materials. First, the amount of Lipofectamine was varied with a fixed amount of DNA or siRNA to determine in which proportions they would form an optimal combination. Finally, to investigate the effect of DNA or siRNA on the co-complexation of both DNA and siRNA, the co-complex of DNA and siRNA was prepared at the various ratios with a fixed amount of DNA. All complexation was confirmed by gel retardation of DNA or siRNA on agarose gels. The effects of siRNA complexes on mRNA expression from plasmid DNA were explored post-transfection, while the influence of plasmid DNA complexes on the transfection of siRNA was determined in GFP-expressing H4IIE cells. The complex between DNA and Lipofectamine was formed at a weight ratio of 0.8:1, whereas the light band of siRNA/Lipofectamine disappeared at a weight ratio of 4:1. When the amounts of DNA, siRNA, and the mixture were fixed, the optimal ratio of nucleic acids and Lipofectamine in our composition was 110:80:350 (ng). Confocal images and flow cytometry showed that inhibition of GFP expression by siRNA was not interfered with by co-complexed plasmid DNA. Moreover, mRNA expression of adiponectin was not hampered by the addition of siRNA; rather, it was increased. Thus, co-complexation of siRNA and plasmid DNA may have a synergistic effect on delivery of the therapeutic gene and siRNA.
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KEYWORD
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siRNA, Plasmid DNA, Lipofectamine, Co-complexation
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