title: One-step encapsulation of siRNA between lipid-layers of multi-layer polycation liposomes by lipoplex freeze-thawing.
abstract: Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCLs) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a "packed multi-layer" structure after the freeze-thawing of "single-layer" PCL and siRNA complex, suggesting that siRNA exists between the lipid layers working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freeze-thawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for "one-step" encapsulation of siRNA into liposomes by freeze-thawing.
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Koide
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Okamoto
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsuchida
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ando
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Ariizumi
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kiyokawa
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Asai
authors.affiliation: Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan.
authors.forename: Takehisa
authors.initials: T
authors.lastname: Dewa
authors.affiliation: Department of Medical Biochemistry, Graduate Division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan. Electronic address: oku@u-shizuoka-ken.ac.jp.
authors.forename: Naoto
authors.initials: N
authors.lastname: Oku
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005615:Freezing; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011092:Polyethylene Glycols / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics; D014162:Transfection
chemical_list: D008055:Lipids; D008081:Liposomes; D011073:Polyamines; D000071228:Polyelectrolytes; D011092:Polyethylene Glycols; D034741:RNA, Small Interfering
keywords: Freeze–thaw; Polycation liposomes; Small-interfering RNA; siRNA encapsulation