Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery

Background One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell-permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS). Methods Two CPP-NLS peptides which differ in the location of the NLS relative to the CPP were used: S4(13)-PV and PV-S4(13). The peptides were attached to pDNA rising electrostatic interactions. Gel-electrophoresis and fluorescent assays were performed to evaluate pDNA-peptide interactions and condensation effects. Confocal microscopy was used to evaluate pDNA-peptide interaction inside cells. Transfection studies were conducted with the luciferase gene, using pDNA-peptides alone, or with the application of TUS. Results Attachment of both peptides to pDNA condensed the pDNA, with higher affinity for the S413-PV peptide. This interaction protected pDNA from endonucleases, but was also reversible. Both peptides mediated pDNA delivery to cell cytoplasm, but less significantly to the nucleus. Thus, both peptides produced transfection in cells, when added after incubation with DNA, with higher transfection-level for PV-S4(13). Application of TUS increased transfection mediated by these peptides, but was not highest compared to transfection using TUS and pDNA alone. Conclusions This Study Suggests that CPP-NLS peptides may be used for condensing pDNA and bringing it into the cell cytoplasm, but their ability to mediate nuclear import of pDNA is insignificant. Copyright (C) 2008 John Wiley & Sons, Ltd.