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Biological effects and cellular uptake of c-myc antisense oligonucleotides and their cationic liposome complexes
T Kanamaru1, T Takagi, Y Takakura
1Department of Drug Delivery Research, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Abstract:
The biological effects and cellular uptake of human c-myc antisense oligonucleotides and their liposome complexes were investigated in vitro using human promonocytic leukemia U937 cells. Antisense phosphorothioate oligonucleotides (S-Oligo) significantly inhibited the growth of U937 cells in a dose-dependent manner. However, no significant effect on cell proliferation was observed with unmodified phosphodiester (P-Oligo) and partially phosphorothioated (PS3-Oligo) oligonucleotides with an antisense sequence and S-Oligo with sense and G-quartet control sequences. In cellular uptake experiments, radiolabeled S-Oligo was taken up by U937 cells more than P-Oligo and PS3-Oligo. Similar results were obtained in mouse peritoneal macrophages used for comparison. Confocal microscopic studies demonstrated a significant distribution of FITC-labeled oligonucleotides on the cell surface and in the cytoplasm in a punctate pattern, but not in the nucleus. When complexed with cationic liposomes, cellular uptake of FITC-labeled P-Oligo or S-Oligo was significantly increased and the fluorescence was located mainly in the nucleus, indicating that the uptake and intracellular pharmacokinetics of both oligonucleotides can be modified by complexation. An inhibitory effect of the complexes was observed at a dose which is ineffective in the case of the oligonucleotides alone. However, this effect was also associated with cytotoxicity of the cationic liposomes, suggesting that optimization of this formulation will be necessary to achieve a more efficient delivery of the oligonucleotides to U937 cells.
Insights
Phosphorothioate antisense oligonucleotides effectively inhibited leukemia cell growth in vitro. Liposome complexation enhanced cellular uptake and nuclear delivery, but cytotoxicity requires further optimization for therapeutic potential.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Antisense oligonucleotides (ASOs) offer targeted gene silencing potential.
- Understanding ASO cellular uptake and biological effects is crucial for therapeutic development.
- Liposome complexation is a strategy to improve nucleic acid delivery.
Purpose of the Study:
- To investigate the in vitro biological effects and cellular uptake of human c-myc antisense oligonucleotides (ASOs) and their liposome complexes.
- To compare the efficacy of different oligonucleotide modifications (phosphorothioate, phosphodiester) and sequences.
- To evaluate the impact of liposome complexation on ASO delivery and intracellular localization.
Main Methods:
- In vitro studies using human promonocytic leukemia U937 cells and mouse peritoneal macrophages.
- Cell proliferation assays to assess growth inhibition.
- Radiolabeling and confocal microscopy to track cellular uptake and localization of FITC-labeled oligonucleotides.
- Liposome complexation with phosphodiester and phosphorothioate oligonucleotides.
Main Results:
- Antisense phosphorothioate oligonucleotides (S-Oligo) significantly inhibited U937 cell growth dose-dependently.
- Unmodified phosphodiester (P-Oligo) and partially phosphorothioated (PS3-Oligo) ASOs showed no significant effect.
- S-Oligo demonstrated higher cellular uptake than P-Oligo and PS3-Oligo in U937 cells and macrophages.
- Liposome complexation significantly increased cellular uptake of both P-Oligo and S-Oligo, with fluorescence predominantly in the nucleus.
- Complexes showed inhibitory effects at lower doses than ASOs alone, but this was linked to liposome cytotoxicity.
Conclusions:
- Phosphorothioate modification enhances the biological activity and cellular uptake of c-myc antisense oligonucleotides.
- Liposome complexation improves ASO delivery and nuclear localization, potentially increasing efficacy.
- Cationic liposomes exhibit cytotoxicity, necessitating formulation optimization for safe and efficient ASO delivery.