Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines

Abdelkader A Metwally, Olivier Reelfs, Charareh Pourzand, Ian S Blagbrough

Research output: Contribution to journalArticle

  • 7 Citations

Abstract

It is important to obtain structure–activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N4,N9-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N4,N9-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N4,N9-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.
LanguageEnglish
Pages1862-1876
Number of pages15
JournalMolecular Pharmaceutics
Volume9
Issue number7
Early online date30 Nov 2011
DOIs
StatusPublished - 2 Jul 2012

Fingerprint

Spermine
Reporter Genes
Small Interfering RNA
Lipids
Gene Silencing
Structure-Activity Relationship
HeLa Cells
Fluorescence Microscopy
Cell Survival
Flow Cytometry
Gene Expression
Cell Line

Cite this

Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines. / Metwally, Abdelkader A; Reelfs, Olivier; Pourzand, Charareh; Blagbrough, Ian S.

In: Molecular Pharmaceutics, Vol. 9, No. 7, 02.07.2012, p. 1862-1876.

Research output: Contribution to journalArticle

@article{4d4285bcbea44fb1b1ecfedc9d2de499,
title = "Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines",
abstract = "It is important to obtain structure–activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N4,N9-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N4,N9-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N4,N9-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.",
author = "Metwally, {Abdelkader A} and Olivier Reelfs and Charareh Pourzand and Blagbrough, {Ian S}",
year = "2012",
month = "7",
day = "2",
doi = "10.1021/mp200429n",
language = "English",
volume = "9",
pages = "1862--1876",
journal = "Molecular Pharmaceutics",
issn = "1543-8384",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines

AU - Metwally,Abdelkader A

AU - Reelfs,Olivier

AU - Pourzand,Charareh

AU - Blagbrough,Ian S

PY - 2012/7/2

Y1 - 2012/7/2

N2 - It is important to obtain structure–activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N4,N9-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N4,N9-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N4,N9-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.

AB - It is important to obtain structure–activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N4,N9-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N4,N9-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N4,N9-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.

UR - http://www.scopus.com/inward/record.url?scp=84863302126&partnerID=8YFLogxK

UR - http://dx.doi.org/10.1021/mp200429n

U2 - 10.1021/mp200429n

DO - 10.1021/mp200429n

M3 - Article

VL - 9

SP - 1862

EP - 1876

JO - Molecular Pharmaceutics

T2 - Molecular Pharmaceutics

JF - Molecular Pharmaceutics

SN - 1543-8384

IS - 7

ER -