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.

doi:10.1021/mp200429n

Cited by 13 publications

(19 citation statements)

References 47 publications

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“…the tetra-amine spermine (1,12-diamino-4,9 diazadodecane), a natural RNA binding agent. [29][30][31] The essential requirements for polynucleotide delivery are set out in detail in some of our recent research papers [32][33][34][35][36] Briefly these are: lipoplex mediated transport of siRNA through the cell membrane and thereby delivery to the cytosol.…”

Section: Introductionmentioning

confidence: 99%

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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

2012

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Our aim is to study the effects of varying the two acyl moieties in synthesized N(4),N(9)-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines, [N(4)-cholesteryloxy-3-carbonyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-stearoyl-, N(4)-lithocholoyl-N(9)-oleoyl-, N(4)-myristoleoyl-N(9)-myristoyl-, and N(4)-oleoyl-N(9)-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 N(4),N(9)-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. N(4)-Oleoyl-N(9)-stearoyl spermine and N(4)-myristoleoyl-N(9)-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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Section: Introductionmentioning

confidence: 99%

“…The rationale for this series of novel asymmetrical cationic lipids is also set out in the previous and following papers. 33,36 We have chosen naturally occurring lipids and covalently bound them in different pairs to the naturally occurring polyamine spermine.…”

Section: Introductionmentioning

confidence: 99%

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

2012

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“…In our research, symmetrical and asymmetrical acyl polyamine derivatives (fatty acid amides of spermine) [152] have been synthesized, characterized, and evaluated as non-viral vectors for siRNA [163,[174][175][176][177]. The intracellular delivery of siRNA and the subsequent sequence specific gene silencing has been quantified by flow cytometry techniques (FACS analysis) [163].…”

Section: Conclusion and Future Avenuesmentioning

confidence: 99%

“…The ability of the spermine conjugates to bind siRNA and form nanoparticles has been investigated and the effect of the complexes of siRNA lipoplexes on the cell viability 48 h posttransfection has been quantified. Our SAR studies allow the identification of the most efficient fatty acids in terms of high gene-silencing efficiency and high cell viability [174][175][176][177][178].…”

Section: Conclusion and Future Avenuesmentioning

confidence: 99%

siRNA and Gene Formulation for Efficient Gene Therapy

Blagbrough¹,

Metwally²

2013

Gene Therapy - Tools and Potential Applications

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Gene Therapy -Tools and Potential Applications 136 plex. This core complex which cζrries-out mRN" degrζdζtion is the RN" induced silencing complex RISC [ -]. The degrζdζtion process requires the key ζrgonζute fζmily of proteins, which contζin ζ domζin with RNζse H endonucleζse type of ζctivity thζt cζtζlyse cleζvζge of the phosphodiester bonds of the tζrgeted mRN". RISC ζssembly ζnd subsequently its function to mediζte sequence specific mRN" degrζdζtion occur in the cytoplζsm of the cell [ ]. The source of the dsRN" segments incorporζted in RISC cζn be endogenously processed microRN" miRN" , short hζirpin RN" shRN" , or synthetic siRN". miRN" is produced from endogenous DN" through the ζction of RN" polymerζse II resulting in the formζtion of non-coding RN" cζlled primζry miRN" pri-miR-N" , which is processed in the nucleus by ζ protein complex contζining ζn enzyme known ζs Droshζ ζnd ζ dsRN" binding protein cofζctor cζlled Pζshζ DGCR . Droshζ cleζves pri-miRN" to produce pre-miRN" , ζ dsRN" of -nucleotides ζnd hζving ζ hζirpin loop, which binds to Exportin protein ζnd is trζnsferred from the nucleus into the cytoplζsm. Pre-miRN" is processed by Dicer RNζse III enzyme in the cytoplζsm to give miRN", typicζlly of nucleotides in length ζnd hζving two nucleotide overhζngs ζt the '-position [ , ], shRN" is produced by trζnscription from ζn exogenous DN" thζt is delivered to the nucleus, ζnd codes for ζ hζirpin shζped RN" with segments of length -nucleotides ζnd loop of nucleotides [ , ] which cζn then be processed by Dicer ζnd incorporζted in the RN"i mζchinery.Once in the cytoplζsm, the processed dsRN" miRN", processed shRN", or siRN" is then incorporζted into ζ protein complex RISC-loζding complex, RLC . In Drosophila the RLC is composed of the dsRN", heterodimer protein DCR Dicer vζriζnt /R D , possibly including the cζtζlytic ζrgonζute proteins ζs well in this complex. The ζctive RISC is formed when one of the RN" strζnds in the complex is cleζved the pζssenger strζnd ζnd the strζnd with the less thermodynζmic stζble '-end guide/ζnti-sense strζnd remζins in the complex. The mRN" with complementζry sequence to the guide strζnd binds to the ζctive RISC ζnd is cleζved by the endoribonucleζse ζctivity of the ζrgonζute component of the complex Figure . . . RNA duplex structure RN" is ζ polymer of ribonucleotides. Eζch RN" nucleotide is composed of one nucleobζse, the monosζcchζride pentose ribose, ζnd one phosphζte group. The nucleobζses in RN" ζre ζdenine purine bζse , guζnine purine bζse , urζcil pyrimidine bζse , ζnd cytosine pyrimidine bζse Figure . " nucleoside is formed when eζch bζse is connected viζ ζ glycosidic bond to the ζnomeric cζrbon ' of ribose, thus when glycosylζted, ζdenine, guζnine, urζcil, ζnd cytosine nucleobζses give ζdenosine, guζnosine, uridine, ζnd cytidine nucleosides. Eζch two nucleosides ζre connected viζ ζ phosphζte diester bond between the ' of one nucleoside ζnd ' of the next nucleoside to form the RN" polynucleotide strζnd. The mζin differences in the primζry structure of RN" ζnd DN" ...

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“…As a result, carbamate-linked lipids have been used in gene delivery systems to achieve high delivery efficiency with low cytotoxicity (16,17). In addition, cationic lipidic molecules with asymmetric dihydrophobic tails were also reported to have possibly superior gene delivery ability (6,(18)(19)(20).…”

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Biotinylated Cyclen‐Contained Cationic Lipids as Non‐Viral Gene Delivery Vectors

Liu

Zhang

et al. 2013

Chem Biol Drug Des

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A series of 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids, namely 5a-c bearing a biotin moiety and a variety of end groups (cholesterol, diosgenin, and α-tocopherol) via biodegradable carbamate bond linkage were prepared and applied as non-viral gene delivery vectors. The liposomes formed from 5 and dioleoylphosphatidylethanolamine could bind and condense plasmid DNA into nanoparticles with appropriate size and zeta potentials. All biotinylated cyclen cationic lipids showed higher cell viability than commercially available lipofectamine 2000 even at high N/P ratios, while their transfection efficiency was relatively lower. Further, results indicate that among the three lipids, α-tocopherol-containing compound 5c has higher DNA-binding ability, lower cytotoxicity, and higher transfection efficiency. Transfection in two different cell lines revealed that these lipoplexes have higher gene delivery efficiency toward tumor cells.

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Efficient Silencing of EGFP Reporter Gene with siRNA Delivered by Asymmetrical N⁴,N⁹-Diacyl Spermines

Cited by 13 publications

References 47 publications

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

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

siRNA and Gene Formulation for Efficient Gene Therapy

Biotinylated Cyclen‐Contained Cationic Lipids as Non‐Viral Gene Delivery Vectors

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Efficient Silencing of EGFP Reporter Gene with siRNA Delivered by Asymmetrical N4,N9-Diacyl Spermines

Cited by 13 publications

References 47 publications

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

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

siRNA and Gene Formulation for Efficient Gene Therapy

Biotinylated Cyclen‐Contained Cationic Lipids as Non‐Viral Gene Delivery Vectors

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Efficient Silencing of EGFP Reporter Gene with siRNA Delivered by Asymmetrical N⁴,N⁹-Diacyl Spermines

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

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