Cells were seeded in 12-well culture plates at a density of 1 x 10^5 per well and allowed to adhere overnight. Cells were transfected with 25, 50 and 100 nM of siRNA using INTERFERin, according to the manufacturer's instructions.

We here report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC) and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) for improved siRNA delivery to cells in vitro and in vivo. Predominantly single walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17 to 34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hundred nm were observed by dynamic light scattering, which potentially might severely limit prolonged in vivo applications. This aggregate formation could be reduced by addition of a cholesterol-hyperbranched polyglycerol surfactant (hbPG) that sterically shields the liposomal surface against serum induced aggregation. In vitro experiments with murine macrophages utilizing macrophage-specific anti-CD68 siRNA loaded liposomes showed potent reduction of CD68 transcript levels without cytotoxicity. Experiments in mice using intravenous application of CW800 NHS ester labeled liposomes, near infrared in vivo imaging and fluorescent assisted cell sorting of inflammatory cells demonstrated an almost quantitative accumulation of these liposomes, with and without hbPG, in the liver and a specific knockdown of CD68 mRNA of up to 70% in liver resident macrophages. Therefore, aggregate formation of TF-liposomes in serum does not significantly affect in vivo siRNA delivery.