Asymmetrical N4,N9-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene

Ian S Blagbrough; Abdelkader A Metwally; Hassan M Ghonaim

doi:10.1021/mp200428d

Asymmetrical N⁴,N⁹-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene

Ian S Blagbrough, Abdelkader A Metwally, Hassan M Ghonaim

Department of Life Sciences

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Abstract

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.

Original language	English
Pages (from-to)	1853-1861
Number of pages	9
Journal	Molecular Pharmaceutics
Volume	9
Issue number	7
Early online date	9 Jan 2012
DOIs	https://doi.org/10.1021/mp200428d
Publication status	Published - 2 Jul 2012

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This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular Pharmaceutics, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/mp200428d
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title = "Asymmetrical N4,N9-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene",

abstract = "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.",

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Asymmetrical N⁴,N⁹-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene

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