Polyethylenimine/oligonucleotide polyplexes investigated by fluorescence resonance energy transfer and fluorescence anisotropy.

To advance knowledge on polyplex structure and composition, fluorescence resonance energy transfer (FRET) and anisotropy measurements were applied to polyplexes of rhodamine-labeled polyethylenimine (PEI) and fluorescein-labeled double-stranded oligodeoxynucleotide (ODN). About 25 kDa PEI was compared with low-molecular-weight PEI of 2.7 kDa. FRET reached maxima at amine to phosphate (N/P) ratios of 2 and 3 for 2.7 kDa and 25 kDa PEI, respectively, with similar average distances between donor and acceptor dye molecules in polyplexes. Anisotropy measurements allowed estimating the bound fractions of PEI and ODN. At N/P = 6, all ODN was bound, but only 58% of PEI 25 kDa and 45% of PEI 2.7 kDa. In conclusion, the higher molecular weight of PEI may conformationally restrict the availability of amino groups for charge interaction with phosphate groups in ODN. Moreover, significant fractions of both types of PEI remain free in solution at N/P ratios frequently used for transfection. FRET and anisotropy measurements provide effective tools for probing polyplex compositions and designing optimized delivery systems.