5296 Nucleic Acids Research, 2011, Vol. 39, No. 12 Finally, we wanted to take our delivery system a step further by developing a suitable formulation for adminis- tration. The formulation process is an indispensable step for the development of any therapeutic product. It has great effect on enhancing the efﬁciency and stability of active pharmaceutical ingredients. Additionally, speciﬁc formulations have to be tailored for different therapeutic approaches. Among the different pharmaceutical forms present, solid formulations remain the most widely used. It is the form used in tablets, capsules, powders for inhal- ation and even powders for injection. This is because they are easy to handle and very stable upon storage and trans- portation. Although CPPs have been extensively exploited in recent years for the delivery of various therapeutics (15), to our knowledge, formulation of CPPs into different pharmaceutical forms has never been thoroughly studied. Therefore, to expand the range of therapeutic applications of CPP-based therapeutics, there is a need for studies exploring the possibility of incorporating CPPs in differ- ent pharmaceutical forms; especially the solid form. To test the feasibility of formulating PF14-SCO complexes into solid formulation, solid dispersion technique was utilized. Although initially invented to increase the solu- bility of poorly water-soluble drugs by dispersing them over hydrophilic solid matrices (33), it is not used for the same purpose in the present work. This is because PF14-SCO complexes are already water-soluble. However, the technique was adopted to enable us obtain uniform distribution of the nanocomplexes over water- soluble excipients by solvent evaporation. For solvent evaporation (in this case water), we chose speed drying under elevated temperature (55–60 C) and reduced pres- sure. Despite being relatively stressful drying method, it is more relevant to the widely used, cost-effective, heat- based pharmaceutical drying techniques. As shown in Figure 6, we found that that lactose-SD 3.33% was the most optimal formulation, achieving splice-correction activity similar to that of the freshly prepared complexes in solution. Mannitol solid dispersions mediated lower activity, while PEG formulations were the least active. It was clear that different excipients and concentrations thereof had a huge impact on the activity of the formula- tion. This suggests that excipients affect the nano- complexes in different ways. The nanocomplexes are formed by non-covalent electrostatic interactions with the ON. Therefore, different excipients varying in their surface charge distribution could interfere with these inter- actions affecting the physicochemical properties of the nanocomplexes and eventually the biological activity. However, an excipient concentration of 3.33% is shown to be relatively superior in all the excipients tested, which suggests that there is an optimal ratio between the concen- tration of nanocomplexes in solution and the amount of solubilized solid material. On the contrary, LF2000 almost lost its entire activity upon application of this drying and dispersion procedure (Figure 6). The success of PF14-SCO complexes to withstand harsh drying and dispersion pro- cedure at which the widely used commercial lipid-based vector failed suggests that PF14-SCO nanocomplexes are physicochemically more stable than the lipoplexes, yet biologically more active. Additionally, we characterized the effect of formulation on the kinetics of splice correc- tion obtained by the best formulation (lactose-SD 3.33%). We found that it demonstrated similar kinetic proﬁle to the freshly prepared nanocomplexes (Supplementary Figure S5). In order to assess that the presence of intact nano- complexes after the formulation procedure, DLS studies were performed comparing the freshly prepared nano- complexes with the solid formulations obtained after drying at 50–60 C . We found that the particles size and particle-size distribution is highly affected by the type of excipient used (Figure 7 and Table 2). Lactose-SD 3.33% formulation, which mediated the highest splice-correction activity, also had DLS proﬁle most similar to the freshly prepared nanocomplexes. This proves that the choice of excipient for formulation of such nanocomplexes is an extremely important step that affects both their physico- chemical properties and biological activity. Yet, another physicochemical property that could affect the biological activity is the zeta potential. Particles of different surface charge are expected to have distinct interactions with the plasma membrane, which should affect the uptake mechanism and efﬁciency of the delivery vector. We expected that a cationic CPP-based nanocomplex should possess net positive surface charge. Surprisingly however, we found that all the nanocomplexes, either freshly prepared or in solid formulation, with or without serum, possessed a net negative charge. The efﬁciency of such nanocomplexes can be explained in light of earlier research on lipoplexes showing that cationic liposome and plasmid DNA complexes are negatively charged under optimal transfection conditions (34) and that this holds in vivo as well (35). In further research studying the uptake mechanisms of CPPs it will be interesting to eluci- date how this net negative charge affects the interaction with the plasma membrane and the biological activity. Next, we subjected the best performing formulation, lactose-SD 3.33%, to stressful stability testing conditions at elevated temperatures for 2 months. Storage at high temperatures increases the rate of degradation reactions that could take years to occur (36). For this reason this method is routinely used to assess the long-term stability of formulations (27,28,37–39). Lactose-SD 3.33% formu- lation demonstrated high stability over the tested time period with no signiﬁcant loss in transfection efﬁciency at most of the time points (Figure 8). The only signiﬁcant decrease in activity was the 60 C 8-weeks time point, where it was 70% of the initial activity. However, this is a remarkably good stability proﬁle for a plain formula- tion without any type of preservatives, coating or special packaging. In addition, these results further corroborate the high stability of PF14 nanocomplexes. In comparison with solid formulation of lipoplexes, PF14 nanocomplexes demonstrate very good activity and stability proﬁles. Earlier reports showing that lipoplexes could be dried over solid matrices mainly utilized the freeze-drying tech- nique in the presence of lyoprotectant sugars like trehal- ose, mannitol or sucrose (38–40). However, despite using sufﬁcient levels of lyoprotectant sugars, signiﬁcant damage was still evident when dilute lipoplex preparations were subjected to freeze-drying (28). This damage occurs