Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery, and the PEG-lipids have been engineered to regulate LNP size and transfection potency. The roles of the other "helper" lipids, DSPC and cholesterol, remain less clear. Here we show that in empty LNP systems that do not contain siRNA, DSPC-cholesterol resides in outer layers, whereas in loaded systems a portion of the DSPC-cholesterol is internalised together with siRNA. It is concluded that the presence of internalised helper lipid is vital to the stable encapsulation of siRNA in the LNP and thus to LNP-siRNA function.