title: Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect.
abstract: Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.
authors.affiliation: Institut für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160 23538, Lübeck, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Veldhoen
authors.forename: Sandra D
authors.initials: SD
authors.lastname: Laufer
authors.forename: Alexander
authors.initials: A
authors.lastname: Trampe
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
mesh_terms: D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
chemical_list: C516249:MPGalpha peptide; D010455:Peptides; D034741:RNA, Small Interfering