## 3 Results and Discussion
### In Vivo Topical Effects  
Typical application of gel formulations containing unloaded SLN and NLC on human volunteers resulted in an increase of skin hydration compared to the control vehicle (gel without lipid nanocarriers, Figure 7). These results were in accordance with previous studies performed on similar formulations and were attributed to the well-known occlusive properties of these lipid nanocarriers [11, 12, 41]. Loading different percentages of IDE into these lipid nanocarriers did not provide any significant increase of skin hydration compared to unloaded SLN and NLC.  
Figure 6: Effect of IDE-loaded SLN and NLC on the vitality percentage of HS68 fibroblast cells exposed to UV radiation: control cells not treated with compounds and not exposed to UV (Co); cells treated with UV radiation alone (UV); cells treated with 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of IDE-loaded SLN or NLC and not exposed to UV (25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL); cells pre-treated with IDE-loaded SLN or NLC (25\(\mathrm{\SIUnitSymbolMicro g}\)/mL) and subsequently exposed to UV radiation (25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL + UV). Each data is presented as mean \(\pm\) SD (n = 12). Different superscript letters indicate a significant difference compared to the control (\(p\) < 0.05).  
Figure 7: Differences in skin hydration values (\(\Delta\) hydration) obtained after a two-week in vivo topical treatment with the gels under investigation. Statistical analysis for the comparison: * \(p\) < 0.05 vs. gel C. Gel C = control gel (without lipid nanocarriers), Gel SLN = gel containing unloaded solid lipid nanoparticles (SLN), Gel NLC = gel containing unloaded nanostructured lipid carriers, Gel SLN IDE1 = gel containing idebenone (IDE) 1% w/w loaded SLN, Gel IDE1.5 = gel containing IDE 1.5 % w/w loaded SLN, Gel NLC IDE1 = gel containing NLC loaded with IDE 1% w/w, Gel NLC IDE1.5 = gel containing NLC loaded with IDE 1.5 % w/w.  
On the contrary, the incorporation of IDE into SLN and NLC provided a decrease in skin pigmentation after treating human volunteers with gels containing such lipid nanocarriers for two weeks (Figure 8). In particular, both IDE-loaded SLN (SLN IDE1 and SLN IDE1.5) led to similar \(\Delta\) pigmentation values that were higher than those obtained from applying the control gel or gels containing unloaded SLN and NLC. A further decrease of skin pigmentation was observed from loading IDE into NLC is likely due to the presence of tocopheryl acetate in these lipid nanocarriers as the photo-protective effects of this vitamin are well-known [42]. Other authors pointed out an increase of photo-protective efficacy in albino mice after loading IDE into NLC [43], thus supporting the hypothesis that NLC could be regarded as suitable carriers for the development of topical formulations with photo-protective activity. In our study, the in vivo topical effects of IDE-loaded SLN and NLC were not dependent on IDE concentration in these lipid nanocarriers. Therefore, we have planned further studies to investigate ID- loaded SLN and NLC interactions with skin tissue and the effects of lipid nanocarrip composition on the photo-protective activity of such nanocarriers.