5292 Nucleic Acids Research, 2011, Vol. 39, No. 12 Figure 4. Uptake of Cy5-labeled SCO, effect of lowering temperature and live cell confocal imaging. (A) HeLa pLuc705 cells (5 104) were seeded 24 h prior to experiments into 24-well plates. Cells were treated with PF14-Cy5-lebeled SCO nanocomplexes at MR5 at three different concentrations for 4 h in serum-free medium followed by the addition of serum to a ﬁnal concentration of 10% and incubated for additional 20 h. For the chloroquine-treated wells, 100 mM chloroquine was added to the complexes prior to cell treatments. Cells were washed with HKR buffer, trypsinized and centrifuged at 1000g. Pellets were lysed in 0.1% Triton X-100 in HKR buffer and ﬂuorescence was measured at 650/670 nm. (i) Fluorescence was recalculated to pmol internalized Cy5 SCO per mg protein. (ii) Luciferase activity was measured for the same cell lysate. The values represent the mean of at least two independent experiments performed in triplicate (mean ± SEM, n = 3). Intergroup differences were considered signiﬁcant at *P < 0.05 using Student’s t-test. (B) HeLa pLuc 705 cells (5 104) were seeded 24 h prior to experiments into 24-well plates. Thirty minutes prior treatment of cells with 50 or100 nM PF14-SCO (MR5) in serum or serum-free media, cells were incubated at 4 or 37 C. Cells were treated for 1 h at indicated temperatures and washed twice with PBS before being placed back at 37 C for additionally 20 h. Cells were processed and analyzed as in the chloroquine experiements. (C) PF14-Cy5-SCO complexes and 10 kDa A488-Dextran were co-incubated with U2OS cells. A major co-localization is seen overall, presented by the appearance of yellow colored vesicles (co-localization of green marked Dextran and red marked PF14-SCO nanocomplexes). The white arrows point out some pre-eminent co-localization vesicles. Several Z-slices per image were taken which allowed us to conﬁrm the co-localization both in X, Y and Z plane in more detail. Such an example is given by the intersection of the green/red (X, Y plane; main picture) and green/blue (Z sectioning panel inserted to the right) or red/blue (Z plane sectioning inserted in the top) lines. The ﬂuorescence imaging was overlaid on top of the differential interference contrast image (DIC) in order to have a relative view of the cell together with the labeled vesicles. stability proﬁle without further additives or special storage conditions. DISCUSSION Several vectors have been utilized to enhance the delivery of ONs. Cationic liposomes have been extensively studied for this purpose; however, despite demonstrating high activity, the associated toxicity has hindered their progress into clinical applications (13). Alternatively, CPPs have been shown to deliver a wide variety of nucleic acid-based therapeutics intracellularly, including SCOs and siRNA (14,29). CPPs covalently conjugated to SCOs have been shown to induce splice-correction activity in different models (7,16). However, this strategy entails laborious chemical conjugation procedures (19). In addition, high doses of the SCO are required to induce signiﬁcant biological response (17). On the other hand, some CPPs have shown to be able to deliver ONs utilizing a non-covalent complexation strategy, especially when chemically modiﬁed with fatty acids (18,20). We have demonstrated earlier that the stearylated versions of TP10 (21) and (RXR)4 (17) show enhanced ability to deliver SCOs and plasmids into cells upon non-covalent