Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell‐penetrating model peptides

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell‐penetrating model peptides of varying helix‐forming propensity and amphipathicity. An HPLC‐based protocol for the synthesis and purification of disulfide bridged conjugates in the 10–100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel‐capillary electrophoresis and laser induced fluorescence detection (GCE‐LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.