Abstract
Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing.
Highlights
IntroductionSiRNA is a synthetic double-stranded (dsRNA) of 21-25 nucleotides per strand. Post-transcriptional gene silencing by siRNA is an important biological tool in functional genomics studies and has many potential therapeutic applications for difficult-to-treat diseases
SiRNA is a synthetic double-stranded of 21-25 nucleotides per strand
During the RNA induced silencing complex (RISC) loading, the passenger siRNA strand is cleaved and only the guide strand is loaded. mRNA possessing a complementary sequence to the guide strand is cleaved by the RISC. siRNA is a potential therapeutic for the treatment of many diseases such as thyroid papillary carcinoma [1] and osteoclast-mediated bone resorption [2], recently reviewed by Blagbrough and Zara [3]
Summary
SiRNA is a synthetic double-stranded (dsRNA) of 21-25 nucleotides per strand. Post-transcriptional gene silencing by siRNA is an important biological tool in functional genomics studies and has many potential therapeutic applications for difficult-to-treat diseases. We have designed a series of symmetrical diacyl lipopolyamines in order to prepare lipoplex formulations (without any pre-preparation of liposomes) of an Alexa Fluor 647-tagged siRNA to investigate if they are suitable for non-toxic transfection of target cells, by forming nanoparticles which will efficiently enter cells for non-viral gene therapy (NVGT) either by endocytosis (a major pathway) or possibly by endocytosis in combination with fusion between siRNA lipoplexes and the plasma membrane (a minor pathway) [9]. Such formulations of lipoplexes lead to RNA knock-down in which the siRNA binding is achieved by anion titration. We provide detailed evidence for the characterization of the nanoparticles, the delivery of Alexa Fluor 647-tagged siRNA and the silencing of GFP biosynthesis, the cell viability, and we compare our results with those obtained with cationic liposomal Lipofectamine 2000 and non-liposomal TransIT TKO
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