Nucleic Acids Research, 2011, Vol. 39, No. 12 5293 Figure 5. Cell conﬂuency dependence and cytotoxicity. (A) HeLa pLuc705 cells seeded at indicated densities 1 day prior to the experiment were treated with 200 nM SCO with either PF14 MR5 or LF2000. (B) Cells were exposed to PF14 (MR5, SCO 200 nM) complexes for 4 h in serum-free medium, followed by addition of serum to a ﬁnal concentration of 10% and incubated for additional 20 h. LF2000 was used according to the manufacturer’s protocol in serum-free conditions with the same SCO dose. Splice-correction assay results are presented as fold-increase untreated cells (control). Toxicity was assessed by Wst-1 assay. The values represent the mean of at least three independent experiments performed in duplicate (mean ± SEM, n = 3). Intergroup differences were considered signiﬁcant at *P < 0.05 using Student’s t-test. complexation. In contrast to other CPPs that are predom- inantly entrapped in endosomal vesicles after internaliza- tion, the remarkably enhanced splice-correction observed with stearyl-TP10 and stearyl-(RXR)4 was mainly due to more efﬁcient endosomal escape (17,21). However, despite being less toxic than lipid-based vectors such as LF2000, these peptides are generally less efﬁcient. Moreover, most transfection reagents are highly active in serum-free con- ditions, but show considerable decrease in their efﬁciency in the presence of serum. This is important, since it immediately suggests that they are less likely to perform well in vivo. In the current work we present a new chemically modiﬁed CPP, PF14. Starting from stearyl-TP10, we utilized ornithines as the main source of positive charges instead of lysines. The design of this peptide was based on earlier reports showing that poly-L-ornithine demon- strated superior transfection efﬁciency (up to 10-fold) compared to equivalent poly-L-lysine-based systems (30). The superior efﬁciency of poly-L-ornithines was related to