With the aim to increase the endosomal escape of siR-NAs after peptide-mediated delivery, Lundberg et al. [101] rationally modified penetratin to form a CPP (termed EB1) with improved endosomolytic properties. They achieved a pH-dependent conformational change of the peptide to a higher degree of helicity by the replacement of two basic amino acids with histidines and the N-terminal addition of six amino acids. In this study, several CPPs were compared in a non-covalent approach by measuring the overall cellular stearyl-R8 n-c EGFP, MAP2B primary rat hippocampal neurons [206] R8-MEND (siRNA/stearyl-R8 core) n-c luciferase HeLa [207] uptake via fluorescence and biological effect of siRNA targeted to luciferase mRNA. Penetratin-as well as TP10mediated transfection did not lead to any silencing of luciferase gene expression, despite high amounts of intracellular siRNA [101] and in contrast to previous reports using siRNA-penetratin-conjugates [90] or TP10/DNA-complexes [102] . EB1-mediated delivery of 100 nM siRNA led to approximately 50 % reduction of luciferase activity. This silencing effect was slightly better than for bPrPp and in the same range as for MPG NLS , but still not as pronounced as for LF2000-mediated transfection of 100 nM siRNA. As it was described earlier, that addition of a pH-sensitive peptide derived from hemagglutinin (HA2) can promote endosomal escape [62] , the authors linked HA2 to penetratin [101] . It turned out that although HA2-penetratin improved the silencing effect when coincubated with penetratin, EB1 was more potent than this combination of peptides. Together with confocal microscopy studies the authors concluded that the lack of biological effect after penetratin-mediated siRNA delivery is due to a lack of endosomal escape and that EB1 has a superior endosomolytic activity in comparison to HA2penetratin.