title: siRNA delivery from triblock copolymer micelles with spatially-ordered compartments of PEG shell, siRNA-loaded intermediate layer, and hydrophobic core.
abstract: Hydrophobized block copolymers have widely been developed for construction of polymeric micelles for stable delivery of nucleic acids as well as anticancer drugs. Herein, we elaborated an A-B-C type of triblock copolymer featuring shell-forming A-segment, nucleic acid-loading B-segment, and stable core-forming C-segment, directed toward construction of a three-layered polymeric micelle as a small interfering RNA (siRNA) vehicle. The triblock copolymer was prepared with nonionic and hydrophilic poly(ethylene glycol) (PEG), cationic poly(l-lysine) (PLys), and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] bearing a hydrophobic dimethoxy nitrobenzyl ester (DN) moiety in the side chain [PEG-PLys-PAsp(DET-DN)]. The resulting triblock copolymers spontaneously formed sub-100 nm-sized polymeric micelles with a hydrophobic PAsp(DET-DN) core as well as PEG shell in an aqueous solution. This micelle was able to incorporate siRNA into the intermediate PLys layer, associated with slightly reduced size and a narrow size distribution. The triblock copolymer micelles (TCMs) stably encapsulated siRNA in serum-containing medium, whereas randomly hydrophobized triblock copolymer [PEG-PLys(DN)-PAsp(DET-DN)] control micelles (RCMs) gradually released siRNA with time and non-PEGylated diblock copolymer [PLys-PAsp(DET-DN)] control micelles (DCMs) immediately formed large aggregates. The TCMs thus induced appreciably stronger sequence-specific gene silencing in cultured cancer cells, compared to those control micelles. The siRNA delivery with TCMs was further examined in terms of cellular uptake and intracellular trafficking. The flow cytometric analysis revealed that the cellular uptake of TCMs was more efficient than that of RCMs, but less efficient than that of DCMs. The intracellular trafficking study using confocal laser scanning microscopy combined with fluorescence resonance energy transfer (FRET) revealed that the TCMs could readily release the siRNA payload within cells, which was in contrast to the DCMs exhibiting much slower release profile. This result indicates that PEG shell contributed to the smooth release of siRNA from TCMs within the cells, presumably due to avoiding irreversible aggregate formation. The obtained results demonstrated that the design of separately functionalized polymer segments expanded the performance of polymeric micelles for successful siRNA delivery.
authors.affiliation: Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kanjiro
authors.initials: K
authors.lastname: Miyata
authors.affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nomoto
authors.affiliation: Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Meng
authors.initials: M
authors.lastname: Zheng
authors.affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Ahram
authors.initials: A
authors.lastname: Kim
authors.affiliation: Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Xueying
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Horacio
authors.initials: H
authors.lastname: Cabral
authors.affiliation: Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: R James
authors.initials: RJ
authors.lastname: Christie
authors.affiliation: Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nishiyama
authors.affiliation: Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Center for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address: kataoka@bmw.t.u-tokyo.ac.jp.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kataoka
mesh_terms: D001593:Benzyl Compounds / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008823:Micelles; D011092:Polyethylene Glycols / Q000737:chemistry*; D011107:Polylysine / Q000031:analogs & derivatives*; D011506:Proteins / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics
chemical_list: D001593:Benzyl Compounds; D008823:Micelles; D011506:Proteins; D034741:RNA, Small Interfering; C571441:poly(ethylene glycol)-poly(N'-(N-(2-aminoethyl)-2-aminoethyl)aspartamide) block copolymer; D011107:Polylysine; D011092:Polyethylene Glycols
keywords: Hydrophobic interaction; Polyion complex micelle; Triblock copolymer; siRNA delivery