Preparation of SNALPs

SNALPs formulations with or without Dox were prepared using ethanol injection method as described elsewhere with slight modifications ²⁴. For in vitro studies, a lipid mixture was prepared from CH: DSPC: DOTAP: C16-PEG2000-Ceramide, with different molar ratios at final lipid amount of 0.213 µmole (Table S1), in absolute ethanol at 60 °C (40 µl). Aqueous solution of siRNA (1 nmole) was diluted with 20 mM citrate buffer (60 µl pH 4, in RNAse free water) and heated at 60 °C. The aqueous siRNA solution was titrated with the alcoholic lipid solution dropwise under vigorous vortex to ensure the formation of SNALPs. The obtained SNALPs were incubated at 40 °C for 1 h. The unentrapped siRNA was removed by ultrafiltration centrifugation using MWCO 100K at 14,000 rpm for 45 min and the prepared SNALPs were re-dispersed in HEPES buffer (to final volume of 100 µl pH 7, in RNAse free water). Different Dox loaded SNALPs were prepared using pH gradient method ²⁵. The SNALPs suspension pH was raised from pH 4 to pH 8 using 0.5 M sodium bicarbonate and heated at 60 °C for 5 min. Subsequently, the SNALPs were incubated with preheated equivolume of Dox solution (in PBS pH 7.4 at drug to lipid ratio 0.2: 1 w/w) at the same temperature for 2 h. Buffer exchange was then performed for all SNALPs (with/or without Dox) using ultrafiltration (MW CO 30K at 14,000 rpm, 45 min) to replace the external buffer with HEPES buffer (pH 7) and to remove the unentrapped payload. Lipid, siRNA and Dox amounts for formulations used in in vivo imaging and therapy studies are summarized in Table S2. For DiR-labeled SNALPs_siNeg used in the biodistribution study, DiR was incorporated in at 1 mole % of total lipid.