# Assessment of the Technological Properties of Idebenone and
## 3 Results and Discussion
It is interesting to note that an increase of nanoparticle mean size was observed for formulations containing TA 3.5% \(w/w\) owing to the incorporation of greater amount of IDE, although no linear relationship could be pointed out. As illustrated in Figure 3, a parabolic relationship was observed between the percentages of TA used to prepare NLC loaded with IDE 1.5% \(w/w\) and the PIT values of the resulting nanoparticles, and the maximum PIT value was observed for nanoparticles containing TA 3% \(w/w\) (formulation VIT 3 IDE 1,5). Conversely, by plotting PIT values against the amount of IDE-loaded into formulations containing TA 3.0 or 3.5% \(w/w\), an almost linear decrease of PIT values was observed by increasing the incorporated amount of IDE (Figure 4; \(\mathbf{r}^{2}\) = 0.969 for VIT3; \(\mathbf{r}^{2}\) = 0.942 for VIT 3,5). These results suggest that formulations containing greater amounts of IDE and TA could be less stable because of their lower PIT values. Figure 3: Relationship between PIT values and% \(w/w\) of tocopherly acetate of NLC loaded with IDE 1.5% \(w/w\). Figure 2: Transmission electron microscopy (TEM) images of (**a**) unloaded SLN and (**b**) unloaded NLC containing decopherly acetate 1% \(w/w\). Stability studies performed storing IDE-loaded lipid nanoparticles for 12 months at room temperature (sheltered from the light) did not highlight any significant change of nanoparticle mean size for formulations containing IDE 1 and 1.5% \(w/w\), regardless of the percentage of TA used to prepare the colloidal dispersion (Figure 5a). The incorporation of a greater amount of IDE led to lower stability of the resulting nanoparticles. In particular, formulations containing IDE percentages greater than 1.5% \(w/w\) gave rise to a slight precipitate after 1-3 months of storage, depending on the TA content (see Figure 5b,c). PDI and \(\zeta\)-potential values did not significantly change for all investigated lipid nanoparticles (data not shown). To give insight into the interactions among IDE and the components of the nanoparticle lipid core, DSC studies were performed on the most stable lipid nanoparticles. Unloaded SLN and NLC were used as a reference to investigate the thermal behavior of IDE-loaded SLN and NLC with long-term stability loading of the highest percentage of IDE 1.5% \(w/w\) (formulations VIT 1 IDE1,5, VIT 2 IDE1,5, VIT 3 IDE 1,5, VIT 3,5 IDE 1,5 and VIT 4 IDE 1,5). In Figure 6, calorimetric curves of SLN and NLC prepared with increasing amount of TA are shown. The calorimetric curve of SLN was characterized by a main peak at 41.90 \({}^{\circ}\)C and a shoulder at a lower temperature. The shoulder could indicate a non-homogenous distribution of the surfactant in the SLN structure. The presence of TA in the nanoparticles produced large variations in the calorimetric curve. NLC VIT 1 showed a main peak at about 40.00 \({}^{\circ}\)C and a shoulder at lower temperature. A single peak was present in the calorimetric curve of NLC VIT 2 but at lower temperature compared to the main peak of NLC VIT 1. Increasing the amount of TA resulted in the shift of the main peak to lower temperatures and in the decrease of its entity up to TA 3.5% \(w/w\), while the calorimetric curve of NLC VIT 4 was an almost flat line, thus suggesting that the lipid core was no longer in the solid state. Calorimetric curves of NLC containing IDE are shown in Figure 7 and compared with those of unloaded and IDE-loaded SLN. The calorimetric curve of SLN IDE showed a main peak at about 41.39 \({}^{\circ}\)C and a shoulder at a higher temperature. These two signals were previously attributed to a prevalent location of IDE at the lipid/surfactant interface [24]. In VIT1 IDE, a main peak at about 40.30 \({}^{\circ}\)C and a shoulder at lower temperature were observed. Increasing the amount of TA caused the shoulder to disappear, whereas the main peak moved to a lower temperature, becoming smaller and smaller until it disappeared almost completely in VIT 3,5 IDE 1,5.