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Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for
1Department of Radiation Medicine, Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007.
Abstract:
We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.
Insights
Phosphorothioate oligonucleotides show better cellular uptake and stability than unmodified ones. Liposome encapsulation further enhances delivery and intracellular transport for potential antisense therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Drug Delivery
Background:
- Achieving effective intracellular delivery of oligonucleotides is crucial for antisense activity.
- Oligonucleotide stability and cellular uptake are key challenges in antisense therapy.
Purpose of the Study:
- To investigate factors affecting intracellular oligonucleotide availability for antisense activity.
- To compare the cellular uptake and intracellular fate of unmodified, phosphorothioate-modified, and liposome-encapsulated oligodeoxynucleotides.
Main Methods:
- Testing 15-20 mer oligodeoxynucleotides (unmodified, phosphorothioate, liposomal) in MOLT-3 leukemia cells.
- Assessing oligonucleotide stability in cell culture medium and intracellular accumulation.
- Utilizing laser-assisted confocal microscopy for intracellular distribution studies.
Main Results:
- Phosphorothioate analogs demonstrated intact cellular penetration and accumulation, unlike unstable unmodified oligomers.
- Intracellular phosphorothioate concentration decreased slowly, mainly due to efflux transport.
- Liposome encapsulation protected oligomers in serum, improved cellular accumulation, and reduced efflux.
- Liposomal delivery facilitated endocytic vesicle release, leading to cytoplasmic and nuclear localization.
Conclusions:
- Phosphorothioate modification enhances oligonucleotide stability and cellular uptake compared to unmodified versions.
- Liposome encapsulation is a promising strategy to improve oligonucleotide delivery, stability, and intracellular transport for antisense applications.
- Understanding intracellular transport mechanisms, including efflux and endocytic pathways, is vital for optimizing oligonucleotide-based therapies.