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Bioorg Med Chem. 2013 Oct 15;21(20):6224-32. doi: 10.1016/j.bmc.2013.04.071. Epub 2013 May 7.

Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs.

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  • 1Laboratory of Chemistry, FDA-CDER, 8800 Rockville Pike, Bethesda, MD 20892, USA.


The bioactivity of a CpG-containing phosphorothioate DNA oligonucleotide with thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) thiophosphate groups in mice led us to investigate the parameters affecting the internalization of these thermosensitive DNA prodrugs in various cell lines. Flow cytometry and confocal microscopy analyses indicate that 5'-fluoresceinated fma-phosphorothioate DNA sequences are poorly internalized in Vero, HeLa and GC-2 cells. However, when four fma-thiophosphate groups of a 15-nucleotide long oligothymidylate prodrug are replaced with 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions, internalization of the positively charged prodrug, under physiological conditions, increased fourfold in HeLa and 40-fold in Vero or GC-2 cells. No cytotoxic effects are observed in Vero cells even at an extracellular prodrug concentration of 50 μM over a period of 72 h. Confocal microscopy studies show that internalization of the positively charged oligothymidylate prodrug in Vero cells is time-dependent with early trafficking of the DNA sequence through endosomal vesicles and, eventually, to the nucleus of the cells. Thus, the incorporation of four 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups into thermosentive fma-phosphorothioate DNA prodrugs is an attractive strategy for efficient cellular internalization of these nucleic acid-based drugs for potential therapeutic indications.

Published by Elsevier Ltd.


Cellular internalization; Nuclear localization; Nucleic acid-based drug delivery; Positively charged DNA prodrug; Thermolytic DNA prodrugs; fma-Thiophosphate protecting group

[PubMed - indexed for MEDLINE]
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