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Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human airway epithelial cells
David de Semir1, Jordi Petriz, Anna Avinyó
1Centre de Genètica Mèdica i Molecular, Institut de Recerca Oncològica, Hospital Duran i Reynals, 08907 L'Hospitalet de Llobregat, Barcelona, Spain.
Background:
Chimeraplasty is a novel methodology that uses chimeric RNA/DNA oligonucleotides (chimeraplasts) to stimulate genomic DNA repair. Efficient uptake and nuclear localization of intact chimeraplasts are key parameters to achieve optimal correction of mutation defects into specific cell types.
Methods:
A 5'-end FITC-labeled 68-mer RNA/DNA oligonucleotide was complexed with the polycation polyethylenimine (PEI) and the cationic lipids Cytofectin and GenePorter. Flow cytometry was employed to evaluate chimeraplast uptake under different conditions. Intracellular chimeraplast distribution and co-localization with endocytosis markers were assessed by confocal microscopy. Relative quantification of chimeraplast metabolism was performed by denaturing PAGE and GeneScan(trade mark) analysis.
Results:
In airway epithelial cells, optimized chimeraplast uptake reached near 100% efficiency with the carriers tested. However, chimeraplast nuclear localization could only be achieved using PEI or Cytofectin. Chimeraplast/GenePorter lipoplexes were retained in the cytoplasm. PEI polyplexes and Cytofectin lipoplexes displayed different uptake rates and internalization mechanisms. Chimeraplast/PEI polyplexes were internalized at least partially by fluid-phase endocytosis. In contrast, phagocytosis may have contributed to the internalization process of large-sized chimeraplast/Cytofectin lipoplexes. Moreover, significant chimeraplast degradation was detected 24 h after transfection with both PEI polyplexes and Cytofectin lipoplexes, although the latter seemed to confer a higher degree of protection against nuclease degradation.
Conclusion:
Both Cytofectin and PEI are efficient for chimeraplast nuclear uptake into airway epithelial cells. However, despite the distinct structures and trafficking pathways of the corresponding complexes, none of them could prevent nuclease-mediated metabolism of the chimeric oligonucleotides. These findings should be taken into account for future investigations of chimeraplast-mediated gene repair in airway epithelial cells.