## 3 Results and Discussion  
### NLCs Preparation and Characterization  
The selection of the components for NLCs preparation is important to obtain good stability of the formulation and high encapsulation efficiency of the extract. For this purpose, solubility studies of SLM in different liquid lipids (Table 1) have been performed. The selected excipients are approved and recognized as safe substance, as the purpose of this study is to produce formulations suitable for oral administration. As evidenced in Table 1, the solubility of SLM in the various analyzed liquid lipids is always greater than in water (0.26 mg/mL), except in the case of borage and hemp oil. The best result was obtained using Capryol 90 (13.80 mg/mL) and Lauroglycol (10.00 mg/mL). Capryol 90 is a colorless liquid with HLB of 5, consisting of mono propylene glycol/esterified with caprylic acid and used for oral, topical, rectal and vaginal formulations. Lauroglycol 90 is an oil with HLB equal to 3, consisting of mono/di-esterified propylene glycol with lauric acid and used as an excipient for topical and oral pharmaceutical formulations. The choice of lipids and surfactants used for NLCs formulation was performed by evaluating not only the solubility of SLM, but also the HLB of oils and tensides, with the aim to obtain good encapsulation efficacy and high stability of the carriers. Stearic acid and Precirol ATO were tested as solid lipids, Capryol and Lauroglycol as liquid lipids. Among the surfactants, selected hydrophilic compounds were Tween 80 (HLB 15), Tween 20 (HLB 16.7) and Brij S20 (HLB 15). These tensides were chosen based on the HLB value and solubilizing capacities. Different ratios of solid and liquid lipids and tensides were evaluated. Some tested formulations showed good physical requires for oral administration, such as size and PDI, but they were not stable. Finally, the optimized NLCs are reported in Table 2. Its composition was Stearic acid/ Brij S20 (1:2 \(w/w\)) and Capryol 90 (2:8 \(w/w\) respect to solid lipid). Brij S20 is a non-ionic tensile, derived from stearic acid covalently grafted with PEG 1000, which form a hydrophilic steric barrier around the NLCs \begin{table}
\begin{tabular}{c c} \hline \hline
**Liquid Lipid** & **SLM Solubility (mg/mL)** \\ \hline Borage oil & 0.12 \(\pm\) 0.01 \\ Capryol 90 & 13.80 \(\pm\) 0.14 \\ Hemp oil & 0.13 \(\pm\) 0.01 \\ Labrafac PG & 0.77 \(\pm\) 0.01 \\ Labrafac WL 1349 & 0.72 \(\pm\) 0.09 \\ Labrafil M 1994 & 2.10 \(\pm\) 0.15 \\ Lauroglycol 90 & 10.00 \(\pm\) 0.01 \\ Oleic acid & - \\ Vitamin E & 0.53 \(\pm\) 0.15 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Solubility of silymarin (SLM) in different liquid lipids/ oils. Results are expressed as means \(\pm\) standard deviation, \(n\) = 3.  
and increase their stability [14]. This selected formulation was able to load 40 mg/mL of extract (SLM-NLCs), with an EE% of 92.4%. The presence of SLM does not influence the stability of the NLCs: when SLM were loaded into the nanoparticles, the formulation showed the same physical parameters than empty NLCs. TEM micrographs provided information on morphology and dimensions of nanoparticles: the formulation had a spherical structure with a diameter like that obtained through DLS analyses (Figure 1). FITC, a lipophilic fluorescent dye, was incorporated into NLCs as a probe to perform in vitro studies with Caco-2 cells and to elucidate trans-endothelial transport. Fluorescent NLCs were prepared as reported for empty NLCs, by adding FITC to the lipid phase (FITC-NLCs). Their physical and chemical parameters are shown in Table 2. All formulations are useful for oral administration. The high \(\zeta\)-potential confirms their stability, as also supported by colloidal stability data. Furthermore, the method applied for the preparation of the NLCs allows avoiding the degradation of the loaded extract or probe, as evidenced by the high recovery values (Table 2).