Considering the multitude of available CPPs, nucleic acid cargos and cellular as well as animal model systems, a comparison of different delivery strategies seems nearly impossible. In this context, we have for the first time undertaken a detailed side by side comparison of two different model systems using the peptide MPG as delivery agent. In the following paragraph we present own experimental data to exemplarily illustrate particular aspects regarding current limitations of peptide-based delivery systems. In contrast to many other CPP procedures, which rely on covalent linkage of carrier and cargo, the peptide MPG forms highly stable non-covalent complexes with nucleic acids, displaying binding constants in the low nanomolar range ( [55] , A. Trampe, unpublished data). The high flexibility of this non-covalent approach can be exploited to easily transport a wide variety of nucleic acid cargos without having to synthesize a new construct for each oligonucleotide. We have used MPG for the delivery of siRNAs in the context of an RNAi-based reporter system and for the delivery of steric block oligonucleotides in the context of the splice correction assay described above [121] . After MPG -mediated transfection of a luciferase-targeted siRNA, we observed strong inhibition of reporter gene readout with an IC 50 in the subnanomolar range [55] . After MPG -mediated transfection of a luciferasetargeted steric block oligonucleotide (further on also referred to as ON-705), we observed a moderate up-regulation of reporter gene readout, representative of low splice correcting activity (Laufer et al., manuscript in preparation). One possible explanation for the different degree of reporter gene regulation could be the different intracellular target sites, i.e. the cytoplasm for siRNAs and the nucleus for splice correction oligonucleotides. To attain more information about the subcellular localization of MPG -oligonucleotide complexes, we performed confocal laser scanning as well as conventional fluorescence microscopy studies with fluorescently labeled siRNAs or steric block oligonucleotides. In both cases, a punctuate non-homogenous distribution of the nucleic acids inside the cells was observed. This pattern is indicative of an accumulation of nucleic acids in endocytotic vesicles, which was verified by coincubation with LysoSensor (Fig. (4A) ). A quantitative computational analysis of fluorescence microscopy data yielded an average of approximately 50 % colocalization between endosomes and siRNA (Fig. (4B) ). In contrast to earlier assumptions that CPPs directly traverse the lipid bilayer, it has commonly become accepted that for most peptide-cargo combinations endocytosis plays a major role in cellular uptake. As described above and in the chapter "Strategies to enhance endosomal escape" below, administration of endosome disruptive substances like chloroquine can greatly increase endosomal release of trapped nucleic acids. Chloroquine is a weak base, non-charged at neutral pH but charged at pH 5.5 [178] . It is able to pass easily through membranes in its uncharged form, but becomes protonated and accumulates within acidic vesicles in its positively charged, membraneimpermeable form. Although its exact mode of action has not yet been resolved, it is generally accepted that chloroquine works via prevention of endosome acidification which in turn increases the residence time of cargo within the endosomes eventually resulting in a higher probability of transfer to the cytoplasm. Fluorescence microscopy analyses in the presence of 100 M chloroquine yielded two quite contrary outcomes. While the localization of siRNA did not change after addition of chloroquine (Fig. (4C) ), for the steric block oligonucleotide the picture changed completely (Fig. (4D) ). In the latter case, a diffuse fluorescence all over the cytoplasm with an accumulation of ON-705 in the nucleus could be observed. Nonetheless, considerable amounts of nucleic acid molecules were still visible as a punctual pattern, which indicates that the chloroquine treatment liberates only a certain fraction. On the whole, these qualitative observations are in full agreement with the observed biologic effects in the absence or presence of chloroquine (Fig. (5A) ). For MPG -mediated transfection of siRNA, even under conditions where the amount of bio-available siRNA was severely limited, only a minor increase in RNAi (ca. 30 %) was measurable. For MPG -mediated transfection of steric block oligonucleotide, on the other hand, a dramatic increase of reporter gene up-regulation by a factor of 50 -100 was observed. However, in both cases, the overall uptake did not change upon incubation with chloroquine ( Fig. (5B) ), which proves that the endosomolytic substance does not interfere with uptake but leads to a re-distribution of internalized nucleic acids. The underlying mechanism for the different effects triggered by chloroquine in case of peptide/siRNA and peptide/steric block oligonucleotide complexes remains unclear. Though, this is a good example that the cargo can substantially affect the properties and thereby intracellular trafficking of a particular carrier system. Ultimately, to assess the overall efficacy of this carrier system, we were interested to elucidate which percentage of molecules taken up after MPG -mediated delivery is biologically active. To derive such information, the exact intracellular amount of intact oligonucleotide, the corresponding reporter signal and the minimal number of molecules necessary to trigger a specific degree of reporter gene modulation have to be known. For the quantification of internalized cargo, we adapted a highly sensitive method first described by Overhoff et al. [176] , enabling us to detect intracellular oligonucleotide amounts down to 10 copies per cell [55] . The method is based on the liquid hybridization of a radioactively labeled probe with the corresponding oligonucleotide in cellular lysates. In this context it should be noted that a stringent heparin wash following the transfection procedure is crucial to avoid an overestimation of intracellular nucleic acid molecules due to complexes attached to the outside of the cell membrane [54, 55] . In order to correlate the numbers derived from the quantification experiments with the minimal number of molecules essential to trigger the observed effect, an independent assay had to be established. The gold standard in this case is microinjection as this technique enables one to deliver definite amounts of nucleic acids with a high degree of bioavailability into the cytoplasm or the nucleus of a mammalian cell along with a low toxicity profile and great accuracy. Considering that after cytoplasmic microinjection only 12 siRNA molecules are sufficient for halfmaximal inhibition of reporter gene expression, one can estimate that of the 10,000 molecules measured after MPGmediated transfection, only ca. 0.1 % are biologically active ( Table 4) . For the splice correction assay, the numbers are different in terms of absolute numbers but the outcome remains the same. Compared to the 300,000 molecules sufficient for maximal splice correction after nuclear microinjection, of the 70,000,000 molecules required following peptide-mediated delivery only ca. 0.5 % are biologically active ( Table 5 ). In both cases >> 99 % of internalized oligonucleotides are most likely retained in endosomes and subsequently degraded in lysosomes after peptide-mediated delivery. Frankly, this is a sobering result and puts in numbers how much room for improvement there actually is. Though it certainly is not legitimate to generalize these findings, there are countless reports in the literature suggesting similar limitations for the majority of non-viral strategies.