title: Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency.
abstract: Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.
authors.affiliation: Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fujimoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.forename: Hiroo
authors.initials: H
authors.lastname: Iwata
mesh_terms: D002460:Cell Line; D004566:Electrodes; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods; D020868:Gene Silencing; D006046:Gold / Q000737:chemistry; D006801:Humans; D011094:Polyethyleneimine / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry; D014162:Transfection / Q000295:instrumentation* / Q000379:methods
chemical_list: D034741:RNA, Small Interfering; D006046:Gold; D011094:Polyethyleneimine