(94 C, 30 s, then 55 C, 30 s, then 72 C, 30 s) for 30 cycles and ﬁnally 72 C, 10 min, for ﬁnal extension. The PCR products were analyzed in a 2% agarose gel in 1 TBE buffer and visualized by SYBR Gold (Invitrogen, Molecular Probes) staining. Gels were documented using the Fluor-S system with a cooled CCD camera (BioRad) and analyzed with the Quantity One software (BioRad). For the mdx mouse myotubes, 24 h after transfec- tion myotubes were washed twice with PBS and total RNA was extracted with 0.5 ml of TRI Reagent (Sigma). RNA preparations were treated with RNase free DNase (2 U) and Proteinase K (20 mg) prior to RT–PCR analysis. RT–PCR was carried out in 12.5 ml with 0.5 mg RNA template using SuperScript III One-Step RT–PCR System with Platinum Taq DNA poly- merase (Invitrogen) primed by forward primer 50-CAG AAT TCT GCC AAT TGC TGAG-30 and reverse primer 50-TTC TTC AGC TTG TGT CAT CC-30. The initial cDNA synthesis was performed at 55 C for 30 min followed by 30 cycles of 95 C for 30 s, 55 C for 1 min and 68 C for 80 s. RT PCR product (1 ml) was then used as the template for secondary PCR performed in 25 ml with 0.5 U Super TAQ polymerase (HT Biotechnologies) and primed by forward primer 50-CCC AGT CTA CCA CCC TAT CAG AGC-30 and reverse primer 50-CCT GCC TTT AAG GCT TCC TT-30. The cycling conditions were 95 C for 1 min, 57 C for 1 min and 72 C for 80 s for 25 cycles. Products were examined by 2% agarose gel and after scanning using Gene Tools Analysis Software (SynGene), the relative amount of exon 23 skipping was expressed as a percentage at a given con- centration of conjugates averaged over duplicates of three experiments. Fluorometry and live-cell confocal imaging HeLa pLuc705 cells (5 104) were seeded 24 h before the experiment in 24-well plates. PF14-Cy5-labeled SCO nanocomplexes (MR5, 200 nM SCO) were added to the cells in 500 ml serum-free medium for 4 h followed by the addition of serum to a ﬁnal concentration of 10% and incubated for additional 20 h. Cells were washed twice in HKR buffer and trypsinized for 10 min with 0.25% trypsin (Invitrogen, Sweden). Cells were then centrifuged at 1000g for 5 min at 4 C, and cell pellets were lysed in 250 ml 0.1% Triton X-100 in HKR buffer for 1 h, after which 200 ml lysate was transferred to a black 96-well plate. Fluorescence was measured at 650/670 nm on FlexStation II ﬂuorescence reader (Molecular devices, USA) and the amount of internalized compound was normalized to the amount of protein was determined using the Lowry method (BioRad, USA). In experiments where splice-correction was measured simultaneously, luciferase activity was measured as mentioned above for the same cell lysate. For live cell confocal imaging micros- copy studies, a number of 3.6 104 U2OS cells were seeded in an 8-well IBItreat m-Slide (IBIDI; LRI instru- ments AB; Stockholm, Sweden), the day before the experi- ment. PF14-Cy5-labeled SCO complexes (MR5, 100nM SCO) and 5 mM of 10 KDa A488-Dextran (Molecular Probes, Invitrogen) were added to the cell medium and Nucleic Acids Research, 2011, Vol. 39, No. 12 5287 co-incubated for 3 h. Cells were then washed extensively with 1 HBSS and ﬁnally 300 ml of OptiMEM without phenol red (Invitrogen) was added. Live cell imaging was then performed using a Zeiss LSM 510 confocal microscope equipped with a Plan-Apochromat 63 /1.4 oil DIC objective with a 0.8 aperture. Images were analyzed and prepared with Zeiss LSM image browser or ImageJ (when imageJ was used the function despeckle was utilized to reduce noise in the image) Cell proliferation Wst-1 Cell proliferation was assessed by the Roche Wst-1 prolif- eration assay according to the manufacturer’s instruc- tions. Brieﬂy, 1 104 cells were seeded 1 day prior to the experiment on a 96-well plate. Cells were treated with PF14-SCO nanocomplexes at different MRs 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. LF2000 was used according to the manufacturer’s protocol in serum-free conditions utilizing the same SCO dose. Wst-1 was added according to the manufacturer’s protocol (Roche Diagnostics Scandinavia AB, Sweden). Wst-1 measures the activity of the mitochondrial dehydro- genases to convert tetrazolium salts to formazan and cell proliferation is directly correlated to the amount of formazan dye that is formed. Absorbance was measured at 450 nm on Digiscan absorbance reader (Labvision via AH Diagnostics AB, Sweden). The percentage of viable cells was determined by normalizing the values obtained for treated cells with untreated cells. Solid dispersion Excipient solutions of lactose monohydrate (Merck Chemicals, Germany), mannitol (Duchefa Biochemie, Holland) and PEG 6000 (Sigma-Aldrich, Sweden) were prepared at ﬁnal concentration of 100 mg/ml in MQ water. PF14-SCO nanocomplexes (MR5, 200 nM) were prepared in 5% of the ﬁnal treatment volume (i.e. 25 ml). After complexation, different proportions of the excipient solutions were added to the ﬁnal concentrations of 1.67, 3.33 and 5% solubilized excipient in the reaction mixture. The mixture was thereafter dried in Savant DNA speed-vac (model DNA 120–230) for 2 h, during which the temperature ranged between 55 and 60 C. Before transfection, the dried powder was reconstituted in 50 ml of MQ water and added to cells grown in 450 ml of serum- containing medium and incubated for 24 h. LF2000 complexes were prepared according to manufacturer’s protocol. Subsequently, the same proportions of the ex- cipient solutions utilized for PF14 formulation were added, and then the mixture was dried and reconstituted similarly. Luciferase activity was measured as stated earlier. Dynamic light scattering and zeta potential studies Hydrodynamic mean diameter of the nanocomplexes was determined by dynamic light scatter (DLS) studies using a Zetasizer Nano ZS apparatus (Malvern Instruments, UK). PF14-SCO nanocomplexes, freshly prepared in solution or in solid dispersion (SD) formulations, were prepared