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

Research output: Contribution to journalArticle

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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.
LanguageEnglish
Pages1853-1861
Number of pages9
JournalMolecular Pharmaceutics
Volume9
Issue number7
Early online date9 Jan 2012
DOIs
StatusPublished - 2 Jul 2012

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Spermine
Reporter Genes
Small Interfering RNA
Fluorescein
Cell Line
Gene Silencing
HeLa Cells
Nanoparticles
Transfection
Steroids
Lipids
Skin
Serum
Neoplasms

Cite this

Asymmetrical N4,N9-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene. / Blagbrough, Ian S; Metwally, Abdelkader A; Ghonaim, Hassan M.

In: Molecular Pharmaceutics, Vol. 9, No. 7, 02.07.2012, p. 1853-1861.

Research output: Contribution to journalArticle

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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.",
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