Efficient and safe delivery of siRNA using anionic lipids: Formulation optimization studies

Novel formulations based on physiologically occurring anionic lipids have been designed to achieve safe and efficient siRNA delivery. Anionic liposomes (DOPG/DOPE) were complexed with siRNA using calcium ion bridges to prepare anionic lipoplexes. Various formulation parameters (liposome composition, lipid and calcium concentration) were evaluated and optimized to achieve efficient silencing and high cell viability in breast cancer cells. The optimal anionic lipoplexes composed of 1μg/mL lipid (40:60 (DOPG/DOPE m/m)), 2.4mM calcium and 10nM siRNA, showed maximum silencing (～70% knockdown) without being cytotoxic. These lipoplexes also showed stability and high efficiency in the presence of serum. Additionally, optimal anionic lipoplexes showed efficient intracellular uptake and endosomal escape. Characterization studies indicated the optimal anionic formulations were 324.2±19.6nm with a surface charge of (-22.9±0.1)mV and 98.5±1.4% encapsulation efficiency. Control cationic lipoplexes (Lipofectamine 2000) showed silencing comparable to the anionic lipoplexes but were highly cytotoxic as indicated by IC50 values (cationic - 22.9μg/mL, compared to anionic - greater than 10(7)μg/mL). Calcium-siRNA complexes (without liposomes) showed low efficiency (～50% silencing), and highly variable results. The optimized anionic formulations may offer a safer alternative to conventional cationic based systems for efficient in vitro as well as in vivo delivery of therapeutic siRNAs.