5290 Nucleic Acids Research, 2011, Vol. 39, No. 12 Figure 2. EC50 and kinetics of PF14 SCO nanocomplexes. (A) The concentration of SCO at which 50% splice-correction occurred is referred to as EC50. HeLa pLuc705 cells were transfected with different concentrations of SCO (25–200 nM) formulated with PF14 at MR5. The control, unrelated ON was used at the highest concentration tested (200 nM). Splice-correction was evaluated by measuring the corrected mRNA levels after running RT–PCR. (i) Splice-correction induced by different concentrations of the SCO. Cells were transfected with PF14 nanocomplexes in either serum-free medium (without serum) or in serum-containing medium (with serum) according to the transfection protocol stated earlier. Splice-correction was calculated as the percentage (percent splice-correction) of the corrected luciferase band to the sum of corrected and aberrant bands. (ii) Levels of corrected mRNA were quantiﬁed in 2% agarose gel using the Quantity One software. Gels are for nanocomplexes with PF14 without serum (top) and PF14 with serum (bottom). Lanes are as follows: 200 nM SCO, 100 nM SCO, 50 nM SCO, 25 nM SCO, 200 nM unrelated SCO (UR) and untreated cells (UT). The upper band in each gel represents the aberrant luciferase mRNA and the lower band is for the corrected form. (B) (i) Kinetics of splice-correction in HeLa pLuc705 cells. The SCO was complexed with PF14 at MR5. Final concentration of the SCO in each well was 200 nM. Transfections were performed in either serum-free (without serum) or serum-containing (with serum) media. (ii) Gels are for complexes with PF14 without serum (top) and PF14 with serum (bottom). Lanes are as follows: 2, 4, 8, 12, 16, 24, 48 h and untreated cells (UT). to variation in cell density compared to LF2000, which progressively lost its activity as the cell number increased. In order to exclude that the high transfection efﬁciency observed with PF14 was associated with increased cyto- toxicity, the activity of mitochondrial dehydrogenases was measured as an indicator of healthy, metabolically active cells using the Wst-1 assay. Even under serum-free condi- tions (Figure 5B), PF14-SCO complexes had no effect on cell viability, whereas LF2000 induced signiﬁcant impair- ment of metabolic activity; a well-known problem of lipid-based ON complexes (lipoplexes) (13). PF14-SCO nanocomplexes can be formulated into solid form by solid dispersion technique To investigate the possibility of formulating PF14-SCO nanocomplexes into solid formulation, we adopted the solid dispersion technique. This technique was used to obtain uniform distribution of the nanocomplexes over hydrophilic solid matrices. Mannitol, lactose and PEG 6000 were used as excipients at various concentrations: 1.67, 3.33 and 5 % solubilized excipient in the reaction mixture (referred to as low, medium and high concentra- tions of excipient, respectively, Figure 6). For drying, we used heating to 55–60 C under vacuum. We found that, after resuspension and addition to the cells, the solid for- mulations were remarkably efﬁcient at inducing splice- correction at rates comparable to the freshly prepared nanocomplexes in solution (Figure 6). Actually, lactose solid dispersion at a concentration of 3.33% (lactose-SD 3.33%) mediated splice-correction activity almost identi- cal to the freshly prepared nanocomplexes. Mannitol solid dispersions displayed slightly lower values, while PEG