Abstract
Purpose
To study the effect of increasing the chain length over C-18 and varying the oxidation level in synthesized N 4,N 9-diacyl spermines on DNA and siRNA formulation, and then to compare their transfection efficiency in cell lines
Methods
The five novel very long chain N 4,N 9-diacyl polyamines: N 4,N 9-[diarachidoyl, diarachidonoyl, dieicosenoyl, dierucoyl and dinervonoyl]-1,12-diamino-4,9-diazadodecane were synthesized. The abilities of these novel compounds to condense DNA and to form nanoparticles were studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with the non-liposomal transfection formulation Lipogen, N 4,N 9-dioleoyl-1,12-diamino-4,9-diazadodecane. Also, the abilities of these compounds to condense siRNA and to form nanoparticles were studied using a RiboGreen intercalation assay and their abilities to deliver siRNA into cells were studied in FEK4 and HtTA cells using fluorescein-labelled Label IT® RNAi Delivery Control, a sequenced 21-mer from Mirus.
Results
We show efficient pEGFP and siRNA formulation and delivery to primary skin and cancer cell lines.
Conclusions
Adding two C20 or C22 chains, both mono-cis-unsaturated, N 4,N 9-dieicosenoyl spermine and N 4,N 9-dierucoyl spermine, gave efficient siRNA delivery vectors, even in the presence of serum, comparable to TransIT-TKO and with excellent cell viability.
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Abbreviations
- EGFP:
-
enhanced green fluorescent protein
- EMEM:
-
Earle’s Minimal Essential Medium
- EthBr:
-
ethidium bromide
- FCS:
-
foetal calf serum
- HRMS:
-
high-resolution mass spectrometry
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- NVGT:
-
non-viral gene therapy
- pEGFP:
-
plasmid encoding for enhanced green fluorescent protein
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Acknowledgements
We acknowledge the financial support of an Egyptian Government studentship to H.M.G. We are grateful to Prof R.M. Tyrrell for the FEK4 and HtTA cell lines and to Dr C. Pourzand (both University of Bath) for helpful advice in cell biology. We thank NanoSight Ltd (Salisbury, UK) for the NanoSight LM10 and Beckman Coulter (High Wycombe, UK) for the Delsa™Nano. We are grateful to M.K. Bates (Mirus Bio Technical Support Team) for the siRNA sequence data.
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Ghonaim, H.M., Li, S. & Blagbrough, I.S. Very Long Chain N 4,N 9 -Diacyl Spermines: Non-Viral Lipopolyamine Vectors for Efficient Plasmid DNA and siRNA Delivery . Pharm Res 26, 19–31 (2009). https://doi.org/10.1007/s11095-008-9705-1
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DOI: https://doi.org/10.1007/s11095-008-9705-1