title: Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.
abstract: Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Hori
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan t-yam@phs.osaka-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Waki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Wada
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Fumito
authors.initials: F
authors.lastname: Wada
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Mio
authors.initials: M
authors.lastname: Noda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan obika@phs.osaka-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D060172:Morpholinos; D053758:Nanoparticles / Q000032:analysis; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D034741:RNA, Small Interfering; D014162:Transfection / Q000379:methods*
chemical_list: D003470:Culture Media; D060172:Morpholinos; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; C477371:locked nucleic acid; D002118:Calcium