title: Impact of plasmid quality on lipoplex-mediated transfection.
abstract: This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, 1049-001, Portugal.
authors.forename: Jonathan
authors.initials: J
authors.lastname: De La Vega
authors.forename: Bas Ter
authors.initials: BT
authors.lastname: Braak
authors.forename: Adriano R
authors.initials: AR
authors.lastname: Azzoni
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
authors.forename: Duarte Miguel F
authors.initials: DM
authors.lastname: Prazeres
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008055:Lipids / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
chemical_list: D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins; D004247:DNA
keywords: downstream processing; gene delivery; gene therapy; lipoplexes; plasmid DNA; purification; transfection