One-step scalable preparation method for non-cationic liposomes with high siRNA content

Cationic liposomes (LPs) have been utilized for short interfering RNA (siRNA) delivery in vitro and in vivo owing to their high affinity for siRNA via electrostatic binding. However, both cytotoxicity and non-specific adsorption of cationic LPs in the body have prevented clinical siRNA applications. These situations have led to siRNA encapsulation in non-cationic LPs. We found that the instillation of neutral phospholipids dissolved in ethanol into aqueous solutions containing siRNA and CaCl2 resulted in high siRNA encapsulation (siRNA encapsulation efficiency: ∼80%; siRNA weight ratio: ∼10 wt% of LPs). The products were monodispersed, ∼200 nm, and negatively charged. Furthermore, when phospholipids with a high-phase transition temperature or cholesterol were used, the encapsulation efficiency and siRNA content remained high. Although anionic LPs could not encapsulate siRNAs using this method, the use of cholesterol-conjugated siRNA helped achieve substantial siRNA encapsulation in anionic LPs. These non-cationic siRNA-containing LPs did not show cytotoxicity in vitro, and could be formed with polyethylene glycol-conjugated phospholipids. When conjugated with targeting ligand, the non-cationic siRNA-containing LPs could suppress the expression of target gene in vitro. These data demonstrate that our preparation method would be suitable for large-scale LP production for systemic siRNA delivery.

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