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Design and Synthesis of N 4,N 9-Disubstituted Spermines for Non-viral siRNA Delivery – Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity

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Abstract

Purpose

To study the effect of sequentially changing the chain length, oxidation level, and charge distribution in N 4,N 9-diacyl and N 4,N 9-dialkyl spermines on siRNA formulation, and then to compare their lipoplex transfection efficiency in cell lines.

Methods

Eight N 4,N 9-diacyl polyamines: N 4,N 9-[didecanoyl, dilauroyl, dimyristoyl, dimyristoleoyl, dipalmitoyl, distearoyl, dioleoyl and diretinoyl]-1,12-diamino-4,9-diazadodecane were synthesized. Their abilities to bind to siRNA and form nanoparticles were studied using a RiboGreen intercalation assay and particle sizing. Two diamides were also reduced to afford tetraamines N 4,N 9-distearyl- and N 4,N 9-dioleyl-1,12-diamino-4,9-diazadodecane. Delivery of fluorescein-labelled Label IT® RNAi Delivery Control was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with TransIT-TKO.

Results

The design, synthesis, and structure-activity relationship studies of a series of N 4,N 9-disubstituted spermines as efficient vectors for non-viral siRNA delivery to primary skin and cancer cell lines is reported. These non-liposomal cationic lipids are promising siRNA carriers based on the naturally occurring polyamine spermine showing that C-18 is a better chain length as shorter chains are more toxic.

Conclusions

N 4,N 9-Distearoyl spermine and N 4,N 9-dioleoyl spermine are efficient siRNA formulation and delivery vectors, even in the presence of serum, comparable to TransIT-TKO. However, four positive charges distributed as in spermine was significantly more toxic.

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Abbreviations

EMEM:

Earle’s Minimal Essential Medium

FCS:

foetal calf serum

HRMS:

high-resolution mass spectrometry

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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Acknowledgements

We acknowledge the financial support of the Egyptian Government to H.M.G. (a fully funded studentship) and to M.K.S. (a studentship under the Channel Scheme). We are grateful to Prof R.M. Tyrrell for the FEK4 and HtTA cell lines, to Dr C. Pourzand for helpful discussions, and we acknowledge S. Li (all University of Bath) for the synthesis of the N 4,N 9-dimyristoleoyl analogue. We thank NanoSight Ltd (Salisbury, UK) for the NanoSight LM10 and Beckman Coulter (High Wycombe, UK) for the Delsa™Nano.

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Authors and Affiliations

  1. Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK

    Moustafa K. Soltan, Hassan M. Ghonaim & Ian S. Blagbrough

  2. Department of Medicinal Chemistry, Zagazig University, Zagazig, Egypt

    Moustafa K. Soltan, Mohamed El Sadek, M. Abou Kull & Lubna Abd El-aziz

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  1. Moustafa K. Soltan
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  2. Hassan M. Ghonaim
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  3. Mohamed El Sadek
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Correspondence to Ian S. Blagbrough.

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Soltan, M.K., Ghonaim, H.M., El Sadek, M. et al. Design and Synthesis of N 4,N 9-Disubstituted Spermines for Non-viral siRNA Delivery – Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity. Pharm Res 26, 286–295 (2009). https://doi.org/10.1007/s11095-008-9731-z

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