Research Paper Design and Synthesis of N 4 ,N 9 -Disubstituted Spermines for Non-viral siRNA Delivery – Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity Moustafa K. Soltan, 1,2 Hassan M. Ghonaim, 1 Mohamed El Sadek, 2 M. Abou Kull, 2 Lubna Abd El-aziz, 2 and Ian S. Blagbrough 1,3 Received July 29, 2008; accepted September 12, 2008; published online October 9, 2008 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® 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. KEY WORDS: lipopolyamines; N 4 ,N 9 -dioleoyl spermine; NVGT; primary skin cells; siRNA delivery. INTRODUCTION The use of an efficient vector for poly-nucleic acid (gene or siRNA) delivery (1) is one of the determining factors for a successful therapy for difficult-to-treat diseases such as cancer (2,3), cystic fibrosis (4,5), and Duchenne muscular dystrophy (6). Among non-viral gene delivery systems, non-liposomal cationic lipids are promising, non-toxic gene carriers. The biological usefulness of spermine (1,12-diamino-4,9- diazadodecane) (7–10) and its conjugates, and the synthesis of the lipopolyamine dioctadecylamidoglycylspermine (DOGS, Transfectam), by Behr et al. (11) as an efficient non-viral transfection agent, encouraged us to focus on the synthesis of novel cationic lipids based on the naturally occurring polyamine spermine (12–15). In an effort to improve the efficiency and control of siRNA delivery, novel spermine based cationic lipid formulations have been investigated within our approach to molecular pharmaceutics for molecular medicine. Methods to deliver poly-nucleic acids fall into two classes. The first employs genetically altered viruses that, in most cases, have had their genome altered or “gutted” to prevent viral replication, reduce cytotoxicity, and permit incorporation of the therapeutic transgene. These vectors can be extremely efficient at producing expression, with essentially only a single viral particle necessary to induce a measurable effect. Problems of low virus titre, an inability to transfect non-dividing cells, induction of strong immune responses, and significant toxicity must still be overcome (16). The second class of delivery systems is collectively referred to as non-viral and involves the use complexes with synthetic carrier molecules e.g. cationic lipids or polymers. Such non-viral delivery systems can potentially have considerable advantages over their viral counterparts e.g. greater control of their molecular composition for simplified manufacturing and analysis (17,18), and relatively lower immunogenicity (5,19– 21), but these non viral systems are still significantly less efficient than viral systems. 0724-8741/09/0200-0286/0 # 2008 Springer Science + Business Media, LLC 286 Pharmaceutical Research, Vol. 26, No. 2, February 2009 ( # 2008) DOI: 10.1007/s11095-008-9731-z 1 Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK. 2 Department of Medicinal Chemistry, Zagazig University, Zagazig, Egypt. 3 To whom correspondence should be addressed. (e-mail: prsisb@ bath.ac.uk) ABBREVIATIONS: EMEM, Earle’s Minimal Essential Medium; FCS, foetal calf serum; HRMS, high-resolution mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.