Effects of cationic lipids in cationic liposomes and disaccharides in the freeze-drying of siRNA lipoplexes on gene silencing in cells by reverse transfection

Abstract

RNA interference is a promising technology to inhibit the production of target proteins, and screening with synthetic small interfering RNA (siRNA) libraries has become a crucial research tool used to study gene function in cells. Reverse (Rev) transfection with freeze-dried siRNA/cationic liposome complexes (siRNA lipoplexes) can simplify and speed up siRNA transfection without the preparation of siRNA lipoplexes just before transfection. In this study, we examined the effects of cationic lipids in cationic liposomes and disaccharides in freeze-drying of siRNA lipoplexes on gene silencing in cells by Rev-transfection. We used three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids for the preparation of cationic liposomes, and we prepared six types of freeze-dried siRNA lipoplexes in the presence of trehalose or sucrose solution in multi-well plates. Increasing concentrations of trehalose or sucrose included during freeze-drying of siRNA lipoplexes resulted in increased gene silencing activity upon Rev-transfection. Strong gene silencing activity was observed regardless of the type of cationic lipid in cationic liposomes when siRNA lipoplexes were freeze-dried with the disaccharides at concentrations of more than 25 mM or 100 mM. In addition, siRNA lipoplexes freeze-dried with 100 mM trehalose or sucrose showed long-term (1 month) stability without apparent loss of gene silencing activity. These findings suggested that Rev-transfection with freeze-dried siRNA lipoplexes may have potential applications in the screening of gene function using siRNA libraries.