title: Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique.
abstract: Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake andin vitrotransfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.
authors.affiliation: Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, 565-8585, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hashimoto
authors.affiliation: Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka, 558-8585, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawazu
authors.affiliation: Silk Materials Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nagasaki
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Murakami
authors.affiliation: Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, 565-8585, Japan.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Yamaoka
keywords: Endosomal escape; Microinjection; Poly(L-arginine); Poly(L-lysine); Transcription efficiency