The poor permeability of the plasma membrane of eukaryotic cells to drugs and DNA, together with the low efficiency of DNA or oligonucleotides to reach their target within cells, constitute the two major barriers for the development of therapeutic molecules. Therefore, over last 10 years, substantial progress has been made in the design of new technologies to improve cellular uptake of therapeutic compounds (Opalinska and Gewirtz, 2002; Järver and Langel, 2004; Glover et al., 2005; Torchilin, 2005; De Fougerolles et al., 2007; Kong and Mooney, 2007). This development has been directly correlated with the dramatic acceleration in the production of new therapeutic molecules. Before then, cell delivery systems were restricted by specific problems. A number of non-viral strategies have been proposed, including lipid-, polycationic-, nanoparticle- and peptide-based methods (Morris et al., 2000; Ogris and Wagner, 2002; Järver and Langel, 2004; Torchilin, 2005), but only a few of these technologies have been efficiently applied in vivo at either pre-clinical or clinical levels. Their major limitations include the poor stability of the complexes and the rapid degradation of the cargo, as well as its insufficient ability to reach its target. CPPs (cell-penetrating peptides) constitute one of the most promising tools for delivering biologically active molecules into cells and therefore play a key role in the future of disease treatments (Järver and Langel, 2004; Joliot and Prochiantz, 2004; Langel, 2007; Moschos et al., 2007). CPPs have been shown to efficiently improve intracellular delivery of various biomolecules, including plasmid DNA, oligonucleotides, siRNA (short interfering RNA), PNA (peptide nucleic acid), proteins and peptides, as well as liposome nanoparticles, into cells both in vivo and in vitro. Short synthetic CPPs have been designed to overcome both extracellular and intracellular limitations, and to trigger the movement of a cargo across the cell membrane into the cytoplasm and improve its intracellular trafficking, thereby facilitating interactions with the target (Gariepy and Kawamura, 2000; Morris et al., 2000; Järver and Langel 2004; Joliot and Prochiantz, 2004;, Deshayes et al., 2005; Snyder and Dowdy, 2005; Langel, 2007). Two major strategies have been described: (1) the covalent linkage of the cargo to the CPP, thereby forming a conjugate which is achieved by either chemical cross-linking, cloning or expression of a protein fused to the CPP (Nagahara et al., 1998; Gait, 2003; Moulton and Moulton, 2004; Zatsepin et al., 2005); and (2) the formation of a non-covalent complex between the two partners. Peptides derived from the trans-activating regulatory protein, TAT, of HIV (Frankel and Pabo, 1998; Fawell et al., 1994; Vives et al., 1997; Schwarze et al., 1999), the third α-helix of Antennapedia homeodomain protein (Derossi et al., 1994; Joliot and Prochiantz; 2004), the VP22 protein from herpes simplex virus (Elliott and O'Hare, 1997), the polyarginine peptide sequence (Wender et al., 2000; Futaki et al., 2001), peptides derived from calcitonin (Schmidt et al., 1998; Krauss et al., 2004) or from antimicrobial peptides buforin I and SynB (Park et al., 2000), as well as polyproline sweet-arrow peptide (Pujals et al., 2006), transportan and derivates (Pooga et al., 1998, 2001; Järver and Langel, 2004) have been successfully used to improve the delivery of covalently linked peptides or proteins into cells and have been shown to be of considerable interest for protein therapeutics (Joliot and Prochiantz, 2004; El-Andaloussi et al., 2005; Murriel and Dowdy, 2006; Langel, 2007). Although conjugation methods offer several advantages for in vivo applications, including rationalization and control of the CPP—cargo, they remain limited from the chemical point of view, as they risk altering the biological activity of the cargoes. In order to offer an alternative to covalent methods, we have proposed a new potent strategy for the delivery of biomolecules into mammalian cells, on the basis of the short amphipathic peptide carriers MPG and Pep (Morris et al., 1997, 2001; Simeoni et al., 2003). MPG and Pep form stable nanoparticles with cargoes without the need for cross-linking or chemical modifications. MPG efficiently delivers nucleic acids (plasmid DNA, oligonucleotides and siRNA) and Pep improves the delivery of proteins and peptides in a fully biologically active form into a variety of cell lines and in vivo (Morris et al., 2001, 2007a, 2007b; Simeoni et al., 2003, 2005). This non-covalent strategy has been recently extended to other CPPs, including TAT (Meade and Dowdy, 2007), polyarginine (Kim et al., 2006; Kumar et al., 2007) and transportan (Lundberg et al., 2007). The mechanism through which MPG and Pep deliver active macromolecules does not involve the endosomal pathway and therefore allows the controlled release of the cargo into the appropriate target subcellular compartment (Deshayes et al., 2004a, 2004b).