Molecular Pharmaceutics, 2012, 9, 1853-1861 Asymmetrical N,N-Diacyl Spermines: SAR Studies of Non-Viral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene

Our aim is to study the effects of varying the two acyl moieties in synthesized N,N-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines: [N-cholesteryloxy-3-carbonyl-N-oleoyl-, N-decanoyl-N-oleoyl-, N-decanoyl-N-stearoyl-, N-lithocholoyl-N-oleoyl-, N-myristoleoyl-N-myristoyl-, and N­ oleoyl-N-stearoyl]-1,12-diamino-4,9-diazadodecane were assessed for their abilities to bind to siRNA, studied using a RiboGreen intercalation assay, and to form nanoparticles. Their siRNA delivery efficiencies were quantified in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA) using a fluorescein-tagged siRNA, and compared with formulations of N,N­ dioleoyl-1,12-diamino-4,9-diazadodecane and of a leading transfecting agent, TransIT-TKO. Transfection was measured in terms of siRNA delivery and silencing of EGFP reporter gene in HeLa cells. By incorporating two different acyl moieties, changing their length and oxidation level in a controlled manner, we show efficient fluorescein-tagged siRNA formulation, delivery, and knock-down of EGFP reporter gene. N-Oleoyl-N-stearoyl spermine and N-myristoleoyl-N­ myristoyl spermine are effective siRNA delivery vectors typically resulting in 89% cell delivery and gene silencing to 34% in the presence of serum, comparable with the results obtained with TransIT-TKO; adding a second lipid chain is better than incorporating a steroid moiety.