Nucleic Acids Research, 2011, Vol. 39, No. 12 5295 Figure 8. Stability testing. Lactose-SD 3.33% formulations were stored either at RT (22 C) or in ovens of adjusted temperatures (40 and 60 C). Samples were monitored for 8 weeks and tested at 0, 2, 4 and 8 weeks. Measurements were carried out in triplicates and differences were considered signiﬁcant at *P < 0.05 according to Student’s t-test. More than 60% splice-correction was achieved as early as 8 h after transfection. This demonstrates that, despite being very stable, the nanocomplexes are rapidly dis- sociated inside the cells releasing SCO cargo. However, natural decline in the signal takes place after 48 h as the nanocomplexes become more dilute due to degradation and cell division (31). In line with the above-mentioned reports (30), our results suggest that ornithine forms very stable nanocomplexes that are more resistant to cellular and serum proteases enabling PF14 to mediate very high levels of splice-correction much more efﬁciently than its predecessor; stearyl TP-10. To demonstrate the activity of PF14 in a disease- relevant model, we used mdx mouse myotubes, a model for DMD. Remarkably, PF14 nanocomplexes were able to induce signiﬁcant levels of splice-correction exceeding LF2000 at MR7 (Figure 3C). Compared to other peptide-based vectors tested in the same model, PF14 is signiﬁcantly more active reaching saturation of the cor- rected band signal at doses ranging from 250 nM for MR5 and 200 nM for MR7, which is several folds lower than doses in the micromolar range used for other vectors (7). Additionally, PF14 effect was not substantially affected in the presence of serum. To quantify the efﬁciency of uptake of SCOs by PF14, ﬂuorescently labeled (Cy5) SCO was used in complex for- mation. The quantitative uptake assay helps to clarify the relation between the amount of the internalized complexes and their biological activity. Interestingly, despite the con- gruency between the uptake data and the splice-correction data in the absence of chloroquine, splice-correction was signiﬁcantly increased in the presence of chloroquine (Figure 4A). We have reported on similar patterns for stearyl-TP10 (21). These data show that, although PF14 nanocomplexes display very efﬁcient splice-correction activity, some of the complexes are entrapped in endosomes and are released after endosomal rupture in the presence of chloroquine. However, despite the increased efﬁciency of PF14 in the presence of chloro- quine, its basal activity levels are much higher than those of stearyl-TP10 and LF2000. This proves that PF14 is a very efﬁcient delivery vector; however, there is space for improvement of its activity by further enhance- ment of endosomal escape. To corroborate the role of endocytosis in the uptake of PF14 nanocomplexes, trans- fections were carried out at lowered temperature (4 C), which is supposed to halt energy-dependent processes like endocytosis. As expected, the transfection efﬁciency of PF14 was markedly decreased at 4 C, indicating the involvement of endocytic processes in the uptake (Figure 4B). To visualize this process ﬂuoresceinyl- labeled dextran, a marker for endocytosis (32), was used. Co-localization between labeled dextran and Cy5-labeled SCO in the cytoplasm strongly suggests a prominent role of endocytosis in the uptake of PF14 nanocomplexes (Figure 4C and Supplementary Figure S3). Further characterization of the efﬁciency of PF14 was done utilizing ﬂow-cytometry to assess the percentage of cells being transfected. Importantly, PF14 was able to transfect nearly 100% of the cell population (Supplementary Figure S4), which explains the nearly 90% correction observed after 24 h with RT–PCR (Figure 2B). Upon testing PF14 over an increasing cell density, it showed less sensitivity than LF2000 towards variations in cell conﬂuency (Figure 5A). This is very im- portant for in vitro screens that require different end- points and for successful in vivo implementation where the target is intact tissues. Comparing the cytotoxicity of both vectors through the activity of mitochondrial dehydrogenases showed that PF14 had no impact on the cell viability and was signiﬁcantly less toxic than LF2000 even in serum-free conditions (Figure 5B).