Asymmetrical N⁴,N⁹-Diacyl Spermines: SAR Studies of Nonviral 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 N4,N9-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines, [N4-cholesteryloxy-3-carbonyl-N9-oleoyl-, N4-decanoyl-N9-oleoyl-, N4-decanoyl-N9-stearoyl-, N4-lithocholoyl-N9-oleoyl-, N4-myristoleoyl-N9-myristoyl-, and N4-oleoyl-N9-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 N4,N9-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. N4-Oleoyl-N9-stearoyl spermine and N4-myristoleoyl-N9-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.