## 3 Results and Discussion
### Caco-2 Cell Culture Experiments  
Permeation studies were performed using a cell-based model in order to complete in vitro characterization of NLCs and understand oral absorption mechanisms. For this aim, Caco-2 cells are considered the most predictive in vitro model to estimate not only passive intestinal diffusion, as previously described for PAMPA, but also active transport processes, paracellular permeability and active efflux [10; 15; 16].  
The cytotoxicity of FITC-NLCs was first investigated by MTS test with the aim to select the most appropriate concentration for transport studies. As shown in Figure 3, NLCs exerted a quite low cytotoxicity (about 30%) diluted 1:180. This is a good result considering that NLCs undergoes a dilution from 1:200 to 1:1000 when they are oral administered.  
Thus, the transport studies were conducted by using this dilution and 1 h as exposition time.  
\(P_{app}\) values are reported in Figure 4; it can be observed that the permeation of FITC, a lipophilic probe not able to cross the Caco-2 monolayer, was improved when formulated into NLCs, reaching an apparent permeability of 102.4 \(\times\) 10\({}^{-6}\) cm/s after 30 min.  
To investigate the possible pathways of FITC-NLCs internalization in Caco-2 cells, its uptake was followed in different energetic conditions and in presence of endocytosis inhibitors.  
The representative images in Figure 5a showed that the internalization of the FITC-NLCs was significantly reduced when incubated at different energetic conditions (4 \({}^{\circ}\)C and ATP-depleted  
Figure 3: Viability of Caco-2 cells exposed to Fluorescein isothioxane-NLCs (FITC-NLCs) (1:20 to 1:180), for 2 h.  
condition). The experimental results indicated in fact that the cellular uptake of NLCs at 4 \({}^{\circ}\)C was only about 37% of that at 37 \({}^{\circ}\)C and sodium azide (an energy inhibitor) was also able to reduce, although to a lesser extent, the cellular uptake. At low temperatures, a higher rigidity and a lower permeability of lipid bilayer have been demonstrated and could explain this difference [45].  
Figure 4: Apparent permeability coefficient (\(P_{app}\)) of free FITC or loaded into NLCs (FITC-NLCs) across the Caco-2 monolayer after 1 h of incubation.  
Figure 5: (a) Cellular uptake of FITC-NLCs (green staining), in the presence of endocytic inhibitors. Nuclei were stained with DAPI. Final magnification 400\(\times\); scale bar 20 \(\upmu\)m. (b) Percentage of intracellular FITC in Caco-2 cells after 1 h of exposure to FITC-NLCs at 37 \({}^{\circ}\)C (100%) or in the presence of endocytic inhibitors. Results are expressed as means \(\pm\) standard deviation, \(n=3\).  
The dependency of cellular uptake on energy indicates that the internalization possibly occurs by endocytosis. To further illustrate about the particular internalization pathway involved, Caco-2 cells were pretreated with inhibitors: chlorpromazine blocks clathrin-dependent endocytosis and indomethacin inhibits caveolin-dependent endocytosis. The cellular uptake of FITC-NLCs seems to be a rather complex process not dependent solely on a single mechanism. Our results indicated that they are incorporated into the intestinal epithelia mainly via the caveolin-dependent endocytic pathway, whereas the endocytosis mediated by clathrin was less involved in the uptake, reducing it by about 20% (Figure 5b).