*2.11. Accessibility of siRNA in siRNA Lipoplexes*

The siRNA association by cationic liposomes was analyzed by exclusion assay using an SYBR® Green I Nucleic Acid Gel Stain (Takara Bio Inc., Shiga, Japan). The siRNA lipoplexes were formed at charge ratios (+:−) of 1:1, 2:1, 3:1, and 4:1. The siRNA lipoplexes with 1 μg of siRNA 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 535 nm with an excitation wavelength of 485 nm using a fluorescence plate reader (ARVO X2, Perkin Elmer, Waltham, MA, USA). As a control, the value of fluorescence obtained upon addition of free siRNA solution was set as 100%. The amount of siRNA available to interact with the SYBR® Green I was expressed as a percentage of the control.
