Cationic switchable lipids: pH-sensitive lipid nanoparticle based on a molecular switch for siRNA delivery

摘要

RNA interference provides a targeted approach for silencing gene expression that may prove beneficial in the treatment of diseases such as cancer and genetic disorders. In order to obtain effective gene knockdown, siRNA's must be entrapped and efficiently conveyed into the cytoplasm of cells. pH-sensitive lipidic nanoparticles improve the cytosolic delivery of genes by supporting endosomal escape after endocytosis. In a previous work we reported the use of switchable lipids that were included into PEGylated liposomal preparations and were able to change conformation upon endosomal acidification (Fig 1A). As a result, a fast and efficient cytoplasmic delivery of a highly polar drug model was observed. We decided to use these properties to develop an efficient siRNA delivery system based on a pH-triggered molecular switch. In this study, we synthesized three new cationic switchable lipids (CSL) that can integrate into the structure of lipid nanoparticles (LNP) (Fig 1B). These CSL's differs by the nature of their cationic headgroup. It is hypothesized that, upon protonation of the switchable lipid that occurs in the endosome after endocytosis, hydrogen-bonding opportunities would favor a conformational change (Fig 1 A) which would disturb the lipid packing of the LNP, inducing fusogenic and destabilization properties, thus conferring endosomal-escape properties. We developed three LNP formulations (Fig 1B). The average diameter of the nanoparticles was ～120 nm. The siRNA's encapsulation efficiency was ～ 93%. We then assayed their transfection efficiencies on a HeLa-GFP model. The CSL1 showed no transfection efficiencies (Fig 2A), whereas the CSL2 and CSL3 showed strong transfection efficiencies (Fig 2B and 2C) without significant cytotoxicity. The intracytoplasmic siRNA delivery of the CSL3-based formulation was assayed by fluorescence microscopy (Fig 2D). Using uptake assays we then characterized the cellular entry of the CSL3-based LNP to provide insight into the mechanism by which the nanoparticles accomplishes cytoplasmic delivery of siRNA. Flow cytometry assays depicting the uptake of CSL3/siRNA-Alexa488 in presence of selective inhibitors of the endocytic pathways helped us to characterize the uptake process. This study indicates that caveolae- and clathrine-mediated endocytosis are the major pathways responsible for CSL3/siRNA uptake (Fig 3A). These findings were also confirmed by fluorescence microscopy using siRNA-Alexa647 (Fig 3A). Following endocytosis, the pH of endosomes and lysosomes is rigorously controlled by acidification via the vacuolar H~+- ATPases. This provides a trigger for the pH-sensitive switchable lipid. Transfection of cells with the CSL3/siRNA was realized in the presence of Baf A1, a known inhibitor of vacuolar H~+ ATPases, to determine if the low pH generated inside the vacuoles is involved in siRNA release from endosomes. Treating cells with 600 nM of Baf A1 led to a complete loss of knockdown efficiency, compared to control cells transfected without Baf A1 (Fig 3B). These data confirm the importance of endosomal acidification in the cytoplasmic release of siRNA when delivered with CSL3/siRNA. We report the use of cationic switchable lipids-based lipid nanoparticle for efficient in vitro delivery of siRNA. The best formulation (CSL3-based) is capable of significant knockdown on a HeLa-GFP model at a siRNA dose as low as 1 nM, without apparent cytotoxicity. This formulation shows much promise for in vivo siRNA delivery to the liver.