A B 60 ,%50 d e ipp 40 k s 3 2n 30 o x e fo 20 t n u om10 A 0 C 50 ,d%40 e p p i sk 30 3 2 n o ex 20 f o t n ou 10 m A 0 in OptiMEM in DMEM/10%FBS LF2000 250 nM 125 nM 62.5 nM 200 nM 100 nM 50 nM in OptiMEM in DMEM/10%FBS LF2000 Figure 3. Splice-correction activity of PF14-SCO nanocomplexes in mdx mouse myotubes. (A) RT–PCR analysis to measure exon skipping activity of PF14-SCO nanocomplexes in mdx mouse myotubes at three different concentrations, at MR5 in serum-free OptiMEM and DMEM/10% FBS in comparison with control LF2000 transfection in OptiMEM. Exon skipping activity was measured as the amount of RT–PCR product after treatment of cells with PF14-SCO nanocomplexes at MR5 (B) or MR7 (C) at three different concentrations. The values represent the mean of at least three independent experiments performed in duplicate (mean ± SEM, n = 3). 6000 solid dispersions demonstrated the highest deviation from the values obtained using the freshly prepared nano- complexes (Figure 6). In contrast, applying the same pro- cedure on LF2000 nearly eliminated any residual activity. On the contrary, the high residual activity of PF14 formu- lations suggests that the PF14 nanocomplexes remain intact after drying and dispersion under these conditions. DLS analysis and zeta potential measurements of PF14-SCO nanocomplexes To conﬁrm the presence of intact nanocomplexes in the formulations, DLS studies and zeta potential measure- ments were performed. These assessments were conducted Nucleic Acids Research, 2011, Vol. 39, No. 12 5291 in the presence of serum to allow possible correlations between such parameters and transfection activity at physiological conditions. Serum contains a number of proteins and protein aggregates that will scatter light re- sulting in a population of particles independent of the PF14-SCO nanocomplexes and can also affect the zeta potential by surface adsorption of some serum proteins. However, we believe that although the presence of serum will affect such measurements, these conditions are more relevant to the physiological ones. For DLS, we have compared the populations detected in serum-containing medium alone and after addition of PF14-SCO nano- complexes. It was possible to distinguish the constant protein aggregates (inherent to the serum, and >100 nm) and a completely independent population associated to the PF14 nanocomplexes (data not shown). Due to the presence of serum proteins, the polydispersity index (PdI) of the solution containing the nanocomplexes does not describe the homogeneity of the PF14 nanocomplexes alone. For this reason, we refer to the shape of the curves that characterize the different populations while eval- uating the homogeneity of the particles. For zeta poten- tial, measurements were carried out either in 0.01 mM KCl or in the presence of 10% serum. After resuspension of the solid formulations, DLS measurements showed that nano- complexes are present in all the tested formulations. However, the uniformity of particles size distribution and the extent of similarity in relation to the freshly prepared nanocomplexes differed with different excipients. Lactose-SD 3.33% formulations were most homoge- neous and most similar to the distribution pattern of the freshly prepared nanocomplexes in solution (Figure 7 and Table 2). These results go in accordance with the transfec- tion results where lactose-SD 3.33% displayed the highest splice-correction activity. Moreover, this analysis demon- strates that different excipients affect the physicochemical characterizes of the nanocomplexes differently in terms of particle size and uniformity, and this effect is reﬂected on the biological activity. We studied the surface charge (zeta potential) of the PF14 nanocomplexes, both freshly prepared and after drying at 50–60 C in the solid disper- sion procedure. Interestingly, independent of formula- tion type and media (presence or absence of serum), the nanocomplexes displayed negative surface charges (Table 2). Solid dispersion formulations are stable for several weeks at elevated temperatures Finally, we tested the stability of the best-performing for- mulation, lactose-SD3.33%, when stored for 2 months at an elevated temperatures. The formulations were stored at three different temperatures (RT, 40 and 60 C) and sampled over an 8 weeks period. We found that the tested formulations were remarkably stable. There was no statistically signiﬁcant loss in transfection efﬁciencies at any time-point except for the 60 C 8-weeks point where the transfection efﬁciency decreased to 70% of the initial value (Figure 8). Compared to lyophilized lipoplexes (27,28), PF14 formulations demonstrated an excellent