Recently, we developed anionic polymer-coated lipoplex of pDNA and found that CS and PGA coatings for cationic lipoplex produced safe systemic vectors [5]. Anionic polymer-coated lipoplexes have already been developed for pDNA delivery; however, there is little information about the use of the anionic polymer-coated lipoplexes for siRNA delivery. Therefore, in this study, we prepared anionic polymer-coated lipoplexes with CS, PGA and poly-aspartic acid (PAA) and examined the biodistribution and gene silencing

effect in the liver after intravenous C646 injection into mice. 1,2-Dioleoyl-3-trimethylammonium-propane methyl sulfate salt (DOTAP) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Poly-l-glutamic acid sodium salt (PGA, 10.5 kDa) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Poly-(α,β)-dl-aspartic acid (PAA, 21 kDa) was obtained from the PolySciTech division of Akina, Inc. (West Lafayette, IN, USA). Cholesterol (Chol) selleck screening library and chondroitin sulfate C sodium salt (CS) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were of the finest grade available. Human breast cancer MCF-7-Luc (TamR-Luc#1) cells stably expressing firefly luciferase (pGL3) were donated by Dr. Kazuhiro Ikeda (Division of Gene Regulation and Signal

Transduction, Research Center Ketotifen for Genomic Medicine, Saitama Medical University, Saitama, Japan) [ 6]. The cells were cultured in Dulbecco’s modified Eagle’s medium

(DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml kanamycin and 0.5 mg/ml G418 at 37 °C in a 5% CO2 humidified atmosphere. siRNAs targeting nucleotides of firefly pGL3 luciferase (Luc siRNA), Cy5.5-labeled Luc siRNA (Cy5.5-siRNA), Luc siRNA conjugated with cholesterol (Luc siRNA-Chol), Cy5.5-labeled Luc siRNA conjugated with cholesterol (Cy5.5-siRNA-Chol), nonsilencing siRNA (Cont siRNA) as a negative control for Luc siRNA, Cont siRNA conjugated with cholesterol (Cont siRNA-Chol) as a negative control for Luc siRNA-Chol, cholesterol-modified apolipoprotein B siRNA (ApoB siRNA-Chol) and Cont siRNA-Chol as a negative control for ApoB siRNA-Chol were synthesized by Sigma Genosys (Tokyo, Japan). The siRNA sequences of the Luc siRNA were as follows: sense strand: 5′-GUGGAUUUCGAGUCGUCUUAA-3′, and antisense strand: 5′-AAGACGACUCGAAAUCCACAU-3. In Cy5.5-siRNA and Cy5.5-siRNA-Chol, Cy5.5 dye was conjugated at the 5′-end of the sense strand, and cholesterol was at the 3′-end of the sense strand. The siRNA sequences of the Cont siRNA were as follows: sense strand: 5′-GUACCGCACGUCAUUCGUAUC-3′, and antisense strand: 5′-UACGAAUGACGUGCGGUACGU-3′ [ 7]. In Luc siRNA-Chol and Cont siRNA-Chol, cholesterol was conjugated at the 3′-end of the sense strand.