Elucidating the encapsulation of short interfering RNA in PEGylated cationic liposomes

Short interfering RNA (siRNA) holds great potential for the treatment of hard-to-cure diseases. One of the major challenges to translate siRNA into drugs is its efficient delivery to its site-of-action, namely the cytoplasm of the target cells. Cationic liposomes have been shown to do the trick, but their short circulation lifetime and potential aggregation in blood limit their applicability for intravenous administration. These hurdles might be overcome by attaching poly(ethylene glycol) (PEG) at the surface of the cationic liposomes through the use of PEGylated lipids. However, this paper reveals that the classical mixing of siRNA with preformed PEGylated cationic liposomes, as frequently done to load PEGylated liposomes with siRNA, prevents an efficient encapsulation of the siRNA in the liposomes. We show that only a minor fraction of the siRNA becomes encapsulated in the core of the PEGylated liposomes, whereas a major part of the siRNA becomes bound at the liposome's outer surface. In serum, the surface-bound siRNA is immediately released and becomes degraded by serum nucleases. By contrast, hydrating a lipid film (containing PEGylated and cationic lipids) directly with a concentrated solution of siRNA (so-called HYDRA protocol), instead of mixing the siRNA with preformed PEGylated liposomes, encapsulates almost 50% of the siRNA in the core of the PEGylated liposomes, which is the maximal encapsulation efficiency for this type of complexes. We show that the siRNA encapsulated in the core of the thus obtained "HYDRA siPLexes" remains fully encapsulated upon dispersing the PEGylated liposomes in human serum.