5294 Nucleic Acids Research, 2011, Vol. 39, No. 12 Figure 6. Solid formulation development and excipient screening. Excipient solutions of lactose, mannitol and PEG 6000 were prepared at ﬁnal concentration of 100 mg/ml in MQ water. PF14-SCO nanocomplexes were prepared in 25 ml MQ water for 1 h, then different proportions of the excipient solutions were added to the ﬁnal concentrations of 1.67% (low), 3.33% (medium) and 5% (high) solubilized excipient in the reaction mixture. The mixture was then dried in speed-vac 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 the wells. LF2000 complexes were prepared according to manufacturer’s protocol. Subsequently, the same proportions of the excipient solutions utilized for PF14 formulation were added, and the mixture was dried and reconstituted similarly. HeLa pLuc705 cells (5 104) were treated with reconstituted complexes for 24 h in serum containing medium. Cells were then lysed in 0.1% Triton X-100 and luciferase activity was measured. The results from splice-correction assay of different formulations of PF14 and LF2000 are presented as fold-increase over untreated cells (control). Figure 7. Dynamic light scattering (DLS). Average diameter of the particles formed by the different formulations studied. Particle size distribution was assessed by dynamic light scatter. All samples were measured in triplicates. The ﬁgure shows representative curves of the populations’ distribution in relation to intensity of the scattered light. All formulations were used at the concentration of 3.33%. the higher afﬁnity for DNA and the ability to make more stable complexes at lower charge ratios (30). Furthermore, we hypothesized that ornithine, being a nonstandard amino acid, would be less prone to serum proteases, and thus could retain the activity in serum conditions. Upon screening for the most optimal MR in the HeLa pLuc705 cell model, we found that MR5 displayed the highest activity in serum-free and serum-containing media (Figure 1). Importantly, in contrast to all tested CPPs so far, PF14 displayed higher activity than LF2000 even in the presence of serum. PF14 activity Table 2. Average diameter of the population of complexes in 0.01 mM KCl or in the presence of 10% serum Samples Average diameter (nm) Zeta Zeta potential (mV) potential (mV) in 0.01mM KCl in OptiMEM 10% FCS Freshly prepared Mannitol Lactose PEG 6000 363.8 ± 52 156.9 ± 43.7 298 ± 57.7 208.3 ± 76 28.4 ± 3.79 15.4 ± 6.31 16.4 ± 4.37 17.7 ± 1.72 12.1 ± 1.41 14.2 ± 1.64 10.4 ± 2.06 9.94 ± 2.55 Average peak is presented in nm (STD). All solid dispersion formula- tions were used at the concentration 3.33%. also exceeded that of OligofectamineTM, RNAiMAXTM and jetPEITM (Supplementary Figure S2). When used at the same concentration, stearyl-TP10 imparted modest activity compared to PF14, which demonstrates the importance of the new modiﬁcations (Figure 1). Also, removal of the stearyl group abolished the activity entirely (Figure 1), emphasizing the role of such a lipo- philic moiety. On the mRNA level, PF14-SCO nano- complexes had a very low EC50 value of 100 nM in both types of media (Figure 2A). Compared to other reported CPPs such as Pip1, RXR4 and R6Pen covalently linked to splice correcting PNA (7), PF14-SCO nano- complexes had at least ﬁve times lower EC50 values. These results add to the evidence that non-covalent com- plexation imparts biological activity at lower ON concen- trations. Kinetics studies showed early onset of activity.