Lipid Nanoparticle (LNP) Encapsulation of RNA

The preparation of LNP and encapsulation of RNA were identical with the previous description (Geall et al., 2012; Ma et al., 2014). Briefly, the LNP, consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Sigma–Aldrich, USA), cholesterol (Sigma–Aldrich), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (PEG DMG 2000) (Avanti Polar Lipids, USA), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) synthesized as reported previously (Heyes et al., 2005) was produced by ethanol dilution process at 10:48:2:40 molar ratio (Geall et al., 2012). Then, the self-amplifying RNA vaccines dissolved in 100 mM citrate buffer were encapsulated in LNP by spontaneous vesicle formation process as previously described (Ma et al., 2014). The resulting RNA/LNP was dialyzed against PBS overnight at 4°C and finally sterile-filtered through a 0.22 μm filter. Prior to vaccination, samples were diluted to the indicated RNA concentration with PBS.