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Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs
Harsh V Jain1, Kazuyo Takeda, Cecilia Tami
1Laboratory of Chemistry, FDA-CDER, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Abstract:
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.
Insights
Researchers improved DNA prodrug delivery by modifying thiophosphate groups. This enhanced cellular uptake significantly, showing promise for nucleic acid-based therapeutics without observed toxicity.
Area of Science:
- Medicinal Chemistry
- Molecular Biology
- Drug Delivery
Background:
- Oligonucleotide therapeutics offer potential but face delivery challenges.
- Thermosensitive DNA prodrugs with specific chemical modifications are being explored.
- Cellular internalization is a critical step for prodrug efficacy.
Purpose of the Study:
- To investigate factors influencing the cellular uptake of thermosensitive DNA prodrugs.
- To evaluate the impact of modifying thiophosphate groups on prodrug internalization.
- To assess the safety and intracellular trafficking of enhanced DNA prodrugs.
Main Methods:
- Flow cytometry and confocal microscopy were used to analyze cellular uptake.
- Synthesis and modification of DNA oligonucleotides with specific thiophosphate groups.
- Cytotoxicity assays were performed on various cell lines (Vero, HeLa, GC-2).
Main Results:
- Standard fma-phosphorothioate DNA sequences showed poor cellular internalization.
- Replacing fma groups with 3-(N,N-dimethylamino)prop-1-yl thiophosphate significantly increased uptake (4-40 fold).
- No cytotoxicity was observed in Vero cells at concentrations up to 50 μM for 72 hours.
Conclusions:
- Incorporating 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups enhances DNA prodrug internalization.
- This modification strategy facilitates efficient cellular uptake and nuclear trafficking.
- Positively charged DNA prodrugs show potential for therapeutic applications in nucleic acid-based drug delivery.
