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 MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-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 MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/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 MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-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.

Despite numerous encouraging reports in the literature, the efficiency of cell penetrating peptides (CPPs) in promoting cellular delivery of bioactive cargos is still limited. To extend our current understanding of the underlying limitations of such approaches, we performed quantitative uptake studies of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides, targeted against a mutated splice site inserted in a firefly luciferase reporter gene construct, applying the peptide carrier MPGalpha as a model system. The peptide formed stable noncovalent complexes with phosphorothioate oligonucleotide (PTO) and locked nucleic acid (LNA) modified oligonucleotides, whereas the neutral peptide nucleic acid (PNA) had to be hybridized to an unmodified DNA to allow for complex formation. Detailed quantitative uptake studies revealed comparable numbers of intracellular PTO and LNA oligonucleotides after peptide-mediated delivery. Surprisingly, the PTO derivative showed the strongest upregulation of reporter gene activity of about 100-fold followed by the PNA (40-fold) and LNA (10-fold). Electroporation and microinjection studies proved that delivery itself was not the limiting factor for the low activity of the LNA derivative. Maximal achievable reporter gene activity could be observed only after addition of chloroquine (CQ), indicative of an endosomal pathway involved. This is in line with nuclear microinjection experiments, which show that the minimal number of steric block molecules needed to trigger the observed reporter upregulation is about two orders of magnitude lower than determined after peptide or cationic lipid delivery.