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Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using
A Islam1, S L Handley, K S Thompson
1Department of Pharmaceutical and Biological Sciences, Pharmaceutical Sciences Institute, Aston University, Birmingham, UK. S.Akhtar@aston.ac.uk
Abstract:
The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.
Insights
Optimized cationic lipoplexes significantly enhance antisense oligodeoxynucleotide (ODN) delivery into C6 glioma cells. These lipoplexes improve cellular association and uptake, directing ODNs to the cytosol and nucleus, bypassing endosomal sequestration.
Area of Science:
- Molecular Biology
- Nanomedicine
- Cell Biology
Background:
- Antisense oligodeoxynucleotides (ODNs) show therapeutic potential but require efficient delivery systems.
- Cationic liposomes and lipoplexes are investigated as carriers for ODNs, yet their intracellular fate is poorly understood.
- Understanding subcellular trafficking is crucial for optimizing ODN delivery and efficacy.
Purpose of the Study:
- To investigate the cellular uptake and biodistribution of ODNs with and without optimized cationic lipoplexes.
- To characterize the intracellular fate and subcellular trafficking of ODN-lipoplex complexes in C6 glioma cells.
- To evaluate the impact of lipoplex formulation on ODN cellular association, distribution, and release.
Main Methods:
- Synthesis of biotin or radiolabeled 15-mer phosphorothioate (PS) ODNs.
- In vitro studies using C6 glioma cell line.
- Characterization of cellular association, cellular efflux, and subcellular biodistribution using Transmission Electron Microscopy (TEM).
Main Results:
- Without lipoplexes, ODNs were primarily sequestered in endosomes and lysosomes, with minimal nuclear and cytoplasmic presence.
- Optimized cationic lipoplexes (DOSPA/DOPE, 3:1 ratio, 1:1 lipid/DNA charge ratio) increased ODN cellular association 10-12 fold.
- Lipoplex delivery resulted in enhanced ODN penetration into the cytosol and nucleus, with reduced vesicular localization and evidence of endosomal escape.
Conclusions:
- Optimized cationic lipoplexes significantly improve cellular delivery of ODNs.
- Lipoplexes favorably modulate ODN intracellular trafficking, promoting entry into non-vesicular compartments like the cytosol and nucleus.
- This enhanced delivery mechanism holds promise for improving the efficacy of antisense oligonucleotide-based therapies.