## 3 Results and Discussion
### CnEO Encapsulation and Release and SLN/CnEO Stability  
The EE of CnEO into SLN was assessed, as previously reported by our group [20]. A UV-Vis scanning of CnEO was performed (230-270 nm), observing a stable and intense peak at 258 nm (Figure S1). Interestingly, CnEO's UV-Vis spectrum and \(\lambda_{\text{max}}\) = 258 nm quite coincided with those of its main component, pulegone [35].. CnEO EE in SLN reached a high value of approximately 97%, which was maintained for at least 6-month storage (Figure 4A). The high amounts of CnEO encapsulated were shown to correlate with those of VOCs such as 1,8-cineole or linalool, previously encapsulated by our group [20; 24].  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **IC50 (\(\mu\)L/L)** & \\ \hline
**Formulation** & **A549** & **HCT-116** \\ \hline SLN/LaDEO & 131 \(\pm\) 8 & 122 \(\pm\) 10 \\ SLN/CnEO & 66 \(\pm\) 5 & 134 \(\pm\) 11 \\ \hline \hline \end{tabular} Dose–response curves were obtained by nonlinear regression, and the IC\({}_{50}\) values were calculated. Data are expressed as the means \(\pm\) SD. Each experiment was carried out in triplicate.  
\end{table}
Table 2: IC50 values of SLN/LaDEO and SLN/CnEO on A549 and HCT-116 cells.  
Figure 2: DLS analysis. (**A**) Particle size distribution, (**B**) Polydispersity index (PDI), and (**C**) _Z_-potential (Z-pot). The results express the mean \(\pm\) SD (n = 3).  
Figure 3: Cytotoxicity of SLN/EO in colon cancer (HCT-116) and lung cancer (A549) cells. HCT-116 (**A**) and A549 (**B**) cells were exposed to increasing concentrations of free and encapsulated EOs or equivalent quantities of empty SLNs (**C**), and cell viability was evaluated by the MTT assay after 24 h. Free EO was dissolved in ethanol (0.1% \(v/v\) max). Results are expressed as means \(\pm\) SD (n = 6) compared to control cells (ethanol 0.1%, 100% viability), (*) \(p\) < 0.001 (70% viability is indicated by the dotted line; below that, the treatment could be considered cytotoxic).  
The kinetic release of CnEO from the SLN matrix was also determined (Figure 4B). To mimic the physiological conditions and the acidic environment found in the endolysosomal compartments, a pH-dependent release of CnEO was tested at pH = 7.4 and 5.0 [21,36]. The free diffusion of CnEO through the dialysis membrane was also evaluated under both conditions. First, it was observed that the SLN/CnEO formulation displayed a conventional bi-phasic release profile at both pHs. An initial robust burst release during the first 4 h was followed by a sustained and slow release for up to 24 h. Then, the CnEO kinetic release from SLN showed a clear pH dependence. At neutral pH, only 43% of the total CnEO was released after 6 h, whereas about 90% was released at acidic pH during the same time. After 24 h, CnEO was almost completely released from SLN at pH = 5.0, whereas only 49% was at pH = 7.4. Additionally, free CnEO was completely released after 4 h at both pHs. Altogether, these findings may explain the reduced hemotoxicity of encapsulated CnEO in comparison with free CnEO at physiological pH. Furthermore, CnEO intracellular release would be favored into acidic endosomes/lysosomes following cellular uptake through the endocytic pathway, described as the primary mechanism of SLN internalization [37].  
To assure the formulation's reproducibility and potential uses, SLN/CnEO stability is a crucial factor. Stability was examined after 6 months of 4 \({}^{\circ}\)C storage in terms of EE (Figure 4A), size, PDI, and Z-pot. No significant changes in the examined parameters were found, as would be expected for this type of system [38, 39, 40], indicating that SLN/CnEO was stable over this period.