Polyethylenimines (PEIs) are synthetic polymers available in branched or linear forms (Figure 2 , upper panels) and in a broad range of molecular weights from < 1000 Da to > 1000 kd. Commercial PEI preparations, although labelled with a defined molecular weight, consist of PEI molecules with a broad molecular weight distribution [45] [46] [47] . PEIs possess a high cationic charge density due to a protonable amino group in every third position [48, 49] . Since no quarternary amino groups are present, the cationic charges are generated by protonation of the amino groups and hence are dependent on the pH in the environment (eg, 20% at pH 7.4, see [50] for review). Due to its ability to condense and compact the DNA into complexes, which form small colloidal particles allowing efficient cellular uptake through endocytosis, PEI has been introduced as a potent DNA transfection reagent in a variety of cell lines and in animals for DNA delivery (for review, see [51, 52] and references therein). In fact, in several studies PEI has been shown to be able to deliver large DNA molecules such as 2.3 Mb yeast artificial chromosomes (YACs) [53] as well as plasmids or small oligonucleotides [48, [54] [55] [56] into mammalian cells in vitro and in vivo. The N/P ratio, which indicates the ratio of the nitrogen atoms of PEI to DNA phosphates in the complex and thus describes the amount of PEI used for complex formation independent of its molecular weight, influences the efficiency of DNA delivery. A positive net charge of the complexes, resulting from high N/P ratios, inhibits due to electrostatic repulsion their aggregation and improves their solubility in aqueous solutions as well as their interaction with the negatively charged extracellular matrix components and thus their cellular uptake [57] . Additionally, the strong buffer capacity, described by the "proton sponge hypothesis" which postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H + buffering polyamines due to enhanced endosomal Cl − accumulation and osmotic swelling/lysis [48] , seems to be responsible for the fact that PEI-based delivery does not require endosome disruptive agents for lysosomal escape. This tight condensation of the DNA molecules as well as the buffering capacity of PEI in certain cellular compartments like endosomes and lysosomes also protects DNA from degradation [48, 49, 58, 59] . PEIs have been successfully used for nonviral gene delivery in vitro and in vivo. While initial publications showed increased transfection efficacies when using high molecular weight PEIs [45] , more recent studies demonstrated the advantages of certain low molecular weight PEIs [47, 60, 61] . The higher transfection efficacy of low molecular weight PEIs may be due to a more efficient uptake of the resulting PEI/DNA complexes, a better intracellular release of the DNA and/or lower in vitro cytotoxicity as compared to high molecular weight PEI [60] [61] [62] [63] . In fact, a decrease in the molecular weight of the PEI leads to an increase in complex size which may be favourable at least for in vitro use [64, 65] . On the other hand, other PEIs with very low molecular weight (< 2 kd) display little or no transfection efficacy even at very high N/P ratios which may be attributed to the fact that a decrease in the molecular weight of PEI has been shown to translate into an increasingly lower ability to form small complexes [63] . Therefore, low molecular weight PEIs require higher N/P ratios for optimal transfection efficacies as compared to higher molecular weight PEIs since higher N/P ratios lead to an increase in compaction with reduced complex sizes and a reduced tendency of the complexes to aggregate due to hydrophobic interactions [61, 63, 64] . Nevertheless, while several parameters have been extensively studied, some precise determinants for transfection efficacy remain to be elucidated (see [50, 66] for review). Also, the mechanism of the cytotoxic effects of PEI complexes is only poorly understood. It may rely on the formation of large aggregates in the range of up to 2 μm which, when formed on the cell surface, impairs membrane functions finally leading to cell necrosis [60] . Clearly, there is a trend towards low molecular weight PEIs as rather nontoxic delivery reagents in vitro and in vivo, which combine high biocompatibility and reduced side-effects thus also allowing to employ larger PEI/DNA complex amounts without significant cytotoxicity. More recently, the use of polyethylenimines has been extended towards the complexation and delivery of RNA molecules, especially small RNA molecules like 37 nt all-RNA ribozymes [67] [68] [69] and siRNAs [70] (Figure 2 ). While chemically unmodified RNA molecules are very instable and prone to rapid degradation, the PEI complexation has been shown to lead to an almost complete protection against enzymatic or nonenzymatic degradation. In fact, PEI-complexed siR-NAs, which are [ 32 P]-labeled for better detection, remain intact in vitro for several hours even in the presence of RNase A or fetal calf serum at 37 • C, while non-complexed siRNAs are rapidly degraded (Figure 3(a) ). This indicates that siRNA molecules are efficiently condensed and thus fully covered and protected by PEI. Indeed, the analysis of PEI/siRNA complexes by atomic force microscopy showed the absence of free siRNAs or siRNA molecule ends and thus confirms these findings regarding an efficient complexation (Grzelinski et al, submitted). However, while the complex stability seems to be sufficient for siRNA protection with all PEIs tested (Werth et al, in press; Aigner et al, unpublished data), several of these complexes do not show any targeting efficacy at all. In fact, only when using certain polyethylenimines, PEI/siRNA complexes are efficiently delivered into target cells in vitro, where siRNAs are released and display bioactivity (Figures 1 and 2) . In general and as seen before for PEI/DNA complexes (see above), the transfection efficacy is dependent on the PEI used, also indicating that the siRNA targeting efficiency mainly depends on the endocytotic uptake of the complex and/or its intracellular decomposition rather than on the in vitro complex stability. Good results were obtained with commercially available JetPEI [70] while the in vivo JetPEI from the same supplier showed only poor siRNA delivery efficacies [71] . Likewise, a novel low molecular weight PEI based on the fractionation of a commercially available polyethylenimine demonstrates high siRNA protection and delivery efficacies in vitro (Werth et al, in press). Under certain conditions, the PEI/RNA (siRNA or ribozyme) complexes retain their physical stability and biological activity also after lyophilization ( [72] and Werth et al, in press). Although the PEI transfection is only transient, data from our lab show that PEI/siRNA effects are stable for at least 7 days (Urban-Klein and Aigner, unpublished results). Finally, another study has explored the use of siRNA nanoplexes comprising of PEI that is PEGylated with an RGD peptide ligand attached at the distal end of the PEI. Again, siRNA nanoplexes protect siRNAs against serum degradation and show in vitro activity [73] .