The siRNA sequences of the Cont siRNA-Chol were as follows : sense strand: 5′-GAACUGUGUGUGAGAGGUCCU*Chol-3′, and antisense strand: 5′-AGGACCUCUCACACACAGUUc*g*C-3′. The lower-case letters represent 2′-O-methyl-modified nucleotides; asterisks represent phosphorothioate linkages. Cationic liposome was prepared from DOTAP/Chol at a molar ratio of 1/1 by a thin-film hydration method, as previously reported [9,10]. For preparation of rhodamine-labeled cationic liposome, Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine-DHPE,
Invitrogen, Carlsbad, CA, USA) was incorporated at 1 mol% into the total lipids. The particle size and ζ-potential of cationic liposomes were measured by dynamic Selisistat purchase light-scattering and electrophoresis light-scattering methods, respectively (ELS-Z2, Otsuka Electronics Co., Ltd., Osaka, Japan). The size of the cationic liposomes was adjusted to approximately 80 nm. To prepare cationic liposome/siRNA complex (cationic lipoplex), cationic liposome suspension was mixed with siRNA by vortex-mixing for 10 s at a charge ratio (−/+) of 1/4, and left for 15 min at room temperature. The theoretical charge ratio (−/+) of siRNA
to cationic liposome was calculated as the molar ratio of siRNA phosphate to DOTAP PCI-32765 solubility dmso nitrogen. To prepare ternary complexes with anionic polymers, cationic lipoplex was mixed with CS, PGA and PAA solutions (CS-, PGA- and PAA-coated lipoplexes, respectively) at the indicated charge ratios. The theoretical charge ratios (−/+) of CS, PGA and PAA to DOTAP were calculated as the molar ratios of sulfate and carboxylic
acid of CS (two negative charges per disaccharide unit), carboxylic acid of PGA (one negative charge per glutamic acid) and carboxylic acid of PAA (one negative charge per aspartic acid) to nitrogen of DOTAP, respectively. After preparation of the cationic lipoplexes, CS-, PGA- and PAA-coated lipoplexes of 1 µg of siRNA or siRNA-Chol at the indicated charge ratios (−/+) of anionic polymer and siRNA to DOTAP, they were analyzed on an 18% acrylamide gel for siRNA in Tris–borate–EDTA (pH 8.0) buffer and were visualized by ethidium bromide staining, as previously reported . siRNA condensation Megestrol Acetate by anionic polymer-coated lipoplexes was analyzed by exclusion assay using an SYBR® Green I Nucleic Acid Gel Stain (Takara Bio Inc., Shiga, Japan), as previously reported . The anionic polymer-coated lipoplexes of 1 µg of siRNA at various charge ratios (−/+) in a volume of 100 µL of Tris–HCl buffer (pH 8.0) were mixed with 100 µL of 2500-fold diluted SYBR® Green I Nucleic Acid Gel Stain solution with Tris–HCl buffer, and then incubated for 30 min. The fluorescence was measured at an emission wavelength of 521 nm with an excitation wavelength of 494 nm using a fluorescent spectrophotometer, F-2700 (Hitachi Co., Ltd., Tokyo, Japan). As a control, the value of fluorescence obtained upon addition of 5 µg/ml free siRNA solution was set as 100%.