Fluorescence resonance energy transfer: Evaluation of the intracellular stability of polyplexes

The investigation of intracellular mechanisms of non-viral nucleic acid delivery systems has provided great impetus for the improvement of their efficacy. Especially the intracellular release of the nucleic acid from the non-viral carrier system may be a relevant criterion for the high transfection efficiency of certain polymers. Therefore, we evaluated fluorescence resonance energy transfer (FRET) in combination with confocal laser scanning microscopy or flow cytometry as tool to determine the intracellular disintegration of polyplexes built with plasmid DNA and linear polyethylenimine. In microscopy, which allowed for an observation of polyplexes within single cells, sensitized emission measurement and acceptor photobleaching have been tested towards quantitative FRET analysis. In contrast, the whole cell population was analyzed by the flow cytometry-based method. We suggest that FRET is a useful tool to evaluate the intracellular disintegration of polyplexes built with various polymers.