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The cell translocation ability of Q4, Q6 and Q8 was quantified by means of flow cytometrywith HeLa, human hepatic Huh-7 cells and human T-lymphocyte Jurkat cells. The resultsindicated that the uptake efficiency remarkably increased with the oligomer size, and whileoctamer Q8 labelled 100% of cells in all cell lines, the affinity of Q4 for all cell lines wasinsignificant. The cellular uptake of Q6 ranged from 26 to 64%, depending on the nature ofthe cell. The higher affinity of the longer oligomers might point to the role of the number ofpositive charges carried by them, as observed in other studies on the cell penetration abilityof cationic peptides and peptoids. The excellent penetration abilities of foldamers Q6 andQ8 was verified by fluorescence microscopy (Figure 2). The punctuated fluorescencepattern observed in the images indicated that the foldamers accumulated in vesicles,suggesting that they follow an endocytic pathway for internalization.In order to investigate the mechanism of translocation, the process was investigated ata low temperature (4 ºC) in Huh-7 cells. At low concentrations (10 and 30 μM), loweringthe temperature substantially reduced the percentage of labeled cells. Intriguingly, at 100μM, 45% and 95% of the cells were still labeled for Q6 and Q8, respectively. However,microscopy fluorescence images showed that the helices appeared to bind strongly to theexternal surface of the plasma membrane, indicating that the high percentage of labeledcells obtained by flow cytometry did not in fact reflect penetration. Such a strong cellmembrane association of the longer helices at a low temperature suggests that thetranslocation is dependent on non-specific, electrostatic interactions of the oligomers withthe membrane. A higher number of positive charges would promote this initial attachment,leading to a more efficient translocation.In conclusion, as part of our investigations on the potential use of aromatic oligoamidefoldamers as drug delivery carriers, we have studied the effect of altering structuralfeatures, such as length and number of charges, on the cell translocation ability offoldamers. The results suggest that a minimum length and number of positive charges isnecessary for efficient cell translocation, with the octamer exhibiting extraordinarypenetration abilities in HeLa, Huh-7 and (more importantly) in the often hard-to-transfectJurkat cell lines. We have also proved that these compounds display practical advantages,including good water solubility and non-toxicity, up to high concentrations. Theexperiments carried out at lower temperatures have provided an insight into the mechanismof the internalization process, suggesting that the uptake of foldamers follows an energydependent pathway. These results have been obtained with non-optimised foldamers.Chemical modification at the side-chain position provides a ready source of diversity andthe possibility to optimise the ability of aromatic foldamers to translocate the plasmamembrane. Further efforts will focus on studying the feasibility of these compounds todeliver bioactive cargo molecules into cells.AcknowledgmentsThis work was supported by ANR contract PCV07_185434, by the European Union (Marie Curie IEF-2009-236605, post-doctoral fellowship to J. I.-A.), by the Government of the Basque Country, and byARC (Post-Doctoral fellowship to K. L.-R.).References1. Hecht, S., Huc, I. Foldamers: Structure, Properties and Applications, Wiley-VCH, Weinheim,2007.2. a) Huc, I. Eur. J. Org. Chem. 1, 17-29 (2004); b) Li, Z.-T., Hou, J.-L., Li, C. Acc. Chem. Res. 41,1343-1353 (2008); c) Gong, B. Acc. Chem. Res. 41, 1376-1386 (2008).3. a) Liu, D., Choi, S., Chen, B., Doerksen, R.J., Clements, D.J., Winkler, J.D., Klein, M.L., DeGrado,W.F. Angew. Chem. Int. Ed. 43, 1158-1162 (2004); b) Tew, G.N., Liu, D., Chen, B., Doerksen, R.J., Kaplan, J., Carroll, P.J., Klein, M.L., DeGrado, W.F. Proc. Natl. Acad. Sci. U.S.A. 99, 5110-5114(2002).4. a) Saraogi, I., Hebda, J.A., Becerril, J., Estroff, L.A., Miranker, A.D., Hamilton, A.D. Angew. Chem.Int. Ed. 49, 736-739 (2010); b) Wilson, A.J. Chem. Soc. Rev. 38, 3289-3300 (2009).5. Iriondo-Alberdi, J., Laxmi-Reddy, K., Bouguerne, B., Staedel, C., Huc, I. ChemBioChem 11, 1679-1685 (2010).6. a) Lv, H., Zhang, S., Wang, B., Cui, S., Yan, J. J. Controlled Release 114, 100-109 (2006).271