title: Novel siRNA delivery system using a ternary polymer complex with strong silencing effect and no cytotoxicity.
abstract: We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.
authors.affiliation: Department of Hospital Pharmacy, Nagasaki University Hospital.
authors.forename: Yukinobu
authors.initials: Y
authors.lastname: Kodama
authors.forename: Yumi
authors.initials: Y
authors.lastname: Shiokawa
authors.forename: Tadahiro
authors.initials: T
authors.lastname: Nakamura
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Kurosaki
authors.forename: Keisei
authors.initials: K
authors.lastname: Aki
authors.forename: Hiroo
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Takahiro
authors.initials: T
authors.lastname: Muro
authors.forename: Takashi
authors.initials: T
authors.lastname: Kitahara
authors.forename: Norihide
authors.initials: N
authors.lastname: Higuchi
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sasaki
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D004912:Erythrocytes / Q000502:physiology; D020868:Gene Silencing; D006386:Hemagglutination Tests; D049409:Luciferases, Firefly / Q000235:genetics; D008297:Male; D051379:Mice; D011094:Polyethyleneimine / Q000737:chemistry*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: D034741:RNA, Small Interfering; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D011094:Polyethyleneimine; D049409:Luciferases, Firefly