Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs

• Published in: Bioorganic & medicinal chemistry Vol. 21; no. 20; pp. 6224 - 6232
• Main Authors: Jain, Harsh V., Takeda, Kazuyo, Tami, Cecilia, Verthelyi, Daniela, Beaucage, Serge L.
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier Ltd 15.10.2013; Elsevier
• Subjects: Animals; Biochemistry & Molecular Biology; Cellular internalization; Cercopithecus aethiops; Chemistry; Chemistry, Medicinal; Chemistry, Organic; cytotoxicity; DNA; DNA - chemistry; DNA - pharmacokinetics; drugs; flow cytometry; Fluoresceins - chemistry; fma-Thiophosphate protecting group; HeLa Cells; Humans; Life Sciences & Biomedicine; Lipids - chemistry; Lipids - pharmacokinetics; Mice; microscopy; Microscopy, Confocal; Nuclear localization; Nucleic acid-based drug delivery; nucleotide sequences; oligonucleotides; Oligonucleotides - chemistry; Oligonucleotides - pharmacokinetics; Pharmacology & Pharmacy; Physical Sciences; Positively charged DNA prodrug; Prodrugs - pharmacokinetics; Science & Technology; Thermolytic DNA prodrugs; Thionucleotides - chemistry; Thionucleotides - pharmacokinetics; thiophosphate; Vero Cells; Thermolytic DNA prodrugs; Nucleic acid-based drug delivery; fma-Thiophosphate protecting group; Nuclear localization; Cellular internalization; Positively charged DNA prodrug; TRANSPORTERS; TRAFFICKING; MECHANISMS; BIOLOGICAL BARRIERS; DELIVERY; LIPOPHILIC PRO-OLIGONUCLEOTIDES; PROTECTING GROUP; HELA-CELLS; ANTISENSE OLIGONUCLEOTIDES; BACTERIAL-DNA
• DOI: 10.1016/j.bmc.2013.04.071
• ISSN: 0968-0896

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 50μM over a period of 72h. 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.