As outlined above, endosomal release is one of the major rate-limiting steps for cellular delivery of macromolecules via cationic lipids, polyplexes and especially CPPs. In the following chapter we will present some examples of how to increase endosomal release. Transfections were performed with 2.1 M MPG and 1 nM siRNA or 10 g/ml LF2000 and 0.02 nM siRNA, i.e. in the range of the IC50 value [55] . Quantification was performed after 24 h according to the liquid hybridization protocol [55] . Molecules per cell were calculated based on the cell number seeded for transfection. For microinjection experiments, molecules per cell were calculated on the basis of the injection volume. Endosome-disrupting substances, like chloroquine, calcium or sucrose, were used to significantly enhance the activity of antisense PNA oligonucleotides conjugated to Tat, oligoarginines or oligolysines [125, 179, 180] . This effect did not result from increased uptake, but rather improved bioavailability in the cytoplasm or nucleus after endosomal escape. Takeuchi et al. [181] showed that by incubation of the target cells with pyrenebutyrate, delivery of arginine-rich peptides could be shifted from endocytic uptake to direct membrane translocation, yielding a rapid distribution of the peptide throughout the cytoplasm, even at 4 °C. Pyrenebutyrate acts as a counteranion and, by interacting with the positively charged peptide, increases the overall hydrophobicity, thereby facilitating a direct translocation through the lipid bilayer, as earlier shown with artificial membranes [182] . This method, which works only in the absence of a medium or serum, was successfully applied for administration of a fluorescent protein and an apoptosis-inducing peptide into dividing as well as non-dividing cells [181] . However, in general the strategies described above are not feasible for in vivo applications, due to high cytotoxicity or other undesirable secondary effects.