Design and Synthesis of N-4,N-9-Disubstituted Spermines for Non-viral siRNA Delivery - Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity

Purpose. To study the effect of sequentially changing the chain length, oxidation level, and charge distribution in N-4,N-9-diacyl and N-4,N-9-dialkyl spermines on siRNA formulation, and then to compare their lipoplex transfection efficiency in cell lines. Methods. Eight N-4,N-9-diacyl polyamines: N-4,N-9-{[}didecanoyl, dilauroyl, dimyristoyl, dimyristoleoyl, dipalmitoyl, distearoyl, dioleoyl and diretinoyl]-1,12-diamino-4,9-diazadodecane were synthesized. Their abilities to bind to siRNA and form nanoparticles were studied using a RiboGreen intercalation assay and particle sizing. Two diamides were also reduced to afford tetraamines N-4,N-9-distearyl- and N-4, N-9-dioleyl-1,12-diamino-4,9-diazadodecane. Delivery of fluorescein-labelled Label IT (R) RNAi Delivery Control was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with TransIT-TKO. Results. The design, synthesis, and structure-activity relationship studies of a series of N-4,N-9-disubstituted spermines as efficient vectors for non-viral siRNA delivery to primary skin and cancer cell lines is reported. These non-liposomal cationic lipids are promising siRNA carriers based on the naturally occurring polyamine spermine showing that C-18 is a better chain length as shorter chains are more toxic. Conclusions. N-4,N-9-Distearoyl spermine and N-4,N-9-dioleoyl spermine are efficient siRNA formulation and delivery vectors, even in the presence of serum, comparable to TransIT-TKO. However, four positive charges distributed as in spermine was significantly more toxic.