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Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with
1Department of Biochemistry, University of Liverpool, UK.
Abstract:
Most mammalian cell types appear to take up antisense oligonucleotides and oligonucleotide analogs from the bathing medium by highly inefficient endocytic mechanisms, and most if not all intracellular oligomer is sequestered in vesicles, still separated by a membrane from the target mRNA. On the other hand, oligonucleotides introduced directly into the cytoplasm by microinjection rapidly accumulate in the cell nucleus. Poor delivery to the designated site of action of antisense oligonucleotides is a major problem limiting their routine use in genetic research and their development as potential therapeutic agents. In view of this difficulty, various means of membrane permeabilization were applied to cultured human leukemia cells in an attempt to enhance intracytoplasmic delivery of fluorescein-tagged oligodeoxynucleotides. The outcome of the manipulations was monitored by flow cytometry and fluorescence microscopy. This work has directly confirmed the conclusion suggested by reported antisense effects, that streptolysin O reversibly permeabilizes the plasma membrane toward oligonucleotides and may be utilized to effect biochemical "microinjection" of these molecules directly into the cytoplasm. KY01 myelogenous leukemia cells treated in this way accumulated over 100-fold higher intracellular levels of oligodeoxynucleotides than in the absence of streptolysin O and, in contrast to the latter case, were observed to concentrate internalized molecules in their nuclei.
Insights
Antisense oligonucleotides face delivery challenges. Streptolysin O enhances cellular uptake by permeabilizing membranes, significantly increasing intracellular levels and nuclear accumulation of these molecules.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Antisense oligonucleotides (ASOs) are limited by inefficient cellular uptake via endocytosis, sequestering them in vesicles away from target mRNA.
- Poor intracellular delivery hinders ASO research applications and therapeutic development.
Purpose of the Study:
- To investigate methods for enhancing the intracytoplasmic delivery of oligodeoxynucleotides into human leukemia cells.
- To evaluate the efficacy of membrane permeabilization techniques for improving ASO cellular uptake.
Main Methods:
- Cultured human leukemia cells were treated with various membrane permeabilization agents.
- Fluorescein-tagged oligodeoxynucleotides were used to track cellular uptake.
- Flow cytometry and fluorescence microscopy were employed to quantify and visualize intracellular oligonucleotide levels.
Main Results:
- Streptolysin O was identified as a reagent that reversibly permeabilizes the plasma membrane to oligonucleotides.
- KY01 myelogenous leukemia cells treated with streptolysin O showed over 100-fold higher intracellular oligodeoxynucleotide levels compared to untreated cells.
- Internalized oligonucleotides concentrated in the nuclei of streptolysin O-treated cells.
Conclusions:
- Streptolysin O facilitates direct cytoplasmic delivery of oligonucleotides, acting as a biochemical microinjection method.
- This approach overcomes endocytic limitations, significantly enhancing intracellular ASO concentrations and nuclear targeting.
- Streptolysin O-mediated delivery holds promise for advancing ASO research and therapeutic applications.