title: Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions.
abstract: The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.
authors.affiliation: Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Itaka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Harada
authors.forename: Kozo
authors.initials: K
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaguchi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kataoka
mesh_terms: D002460:Cell Line; D004355:Drug Stability; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors / Q000737:chemistry*; D006801:Humans; D008055:Lipids; D008823:Micelles; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D011092:Polyethylene Glycols; D011107:Polylysine; D014162:Transfection
chemical_list: C099578:(O-methylpoly(ethylene glycol)-O'-succinyl)-N-epsilon-poly(L-lysine); D008055:Lipids; C086724:Lipofectamine; D008823:Micelles; D011107:Polylysine; D011092:Polyethylene Glycols