Effect of pre­freezing and saccharide types in freeze­drying of siRNA lipoplexes on gene­silencing effects in the cells by reverse transfection.

Our previous study reported that reverse (Rev)­transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) freeze­dried in trehalose or sucrose solution resulted in high gene­silencing activity in cells. The current study investigated whether pre­freezing or saccharide types present during the freeze­drying of siRNA lipoplexes affected gene­silencing in cells after Rev­transfection. Three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids were used for the preparation of cationic liposomes. Additionally, six types of siRNA lipoplexes were vacuum­dried in trehalose or sucrose solution without a pre-freezing process in multi­well plates. A strong gene­silencing activity after Rev­transfection was observed regardless of the cationic lipid types in the cationic liposomes. It was also investigated whether saccharide types in the freeze­drying of siRNA lipoplexes affected gene­silencing after Rev­transfection. siRNA lipoplexes freeze­dried in monosaccharides (glucose, fructose, galactose or mannose), disaccharides (maltose, lactose, lactulose or cellobiose) and trisaccharide solution (raffinose or melezitose) demonstrated high gene­silencing activity. However, following Rev­transfection with siRNA lipoplexes freeze­dried in monosaccharides or trisaccharides, certain saccharides induced cytotoxicity and/or off­target effects. The results of the current study indicated that disaccharides may be suitable for the preparation of vacuum­dried or freeze­dried siRNA lipoplexes for Rev­transfection.

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

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.