## 3 Results and Discussion
### Anticancer Mechanisms of CnEO and SLN/CnEO  
Essential oils and their major VOCs, monoterepenes, were shown to exert cytostatic, cytotoxic, and antimetastatic effects, leading to the inhibition of cancer cell progression [6; 44]. One of the main mechanisms by which EO and conventional anticancer drugs induce cell death depends on their ability to depolarize the mitochondrial membrane, leading to apoptosis and/or autophagic cell death [6]. In addition, the permeabilization of the mitochondrial membrane caused by the convergence of numerous death signal transduction pathways results in the release of pro-apoptotic factors into the cytosol, such as cytochrome C, triggering apoptotic cell death [45]. We have previously shown that mitochondrial membrane depolarization is one of the earliest events involved in monoterepene-induced cell cycle arrest and/or cell death in A549 [26] and other cancer cell types [46; 47]. Here, we evaluated A549 cell death and mitochondrial membrane depolarization after exposure to  
Figure 5: Biocompatibility of SLN/CnEO. (**A,B**) Hematoxicity of empty SLN, free CnEO, and SLN/CnEO. Hematoxicity of 100, 200, and 400 \(\mu\)L/L CnEO and equivalent doses of empty SLN and SLN/CnEO were evaluated after 24 h (**A**) and 48 h (**B**) of exposure. Results are expressed as the mean \(\pm\) SD (n = 3). (**C**) (\(-\)): untreated; (**C**) (\(+\)): 1% Triton X-100-lysed erythrocytes. (**C**) Effect of SLN/CnEO on normal lung WI-38 fibroblasts. Cells were exposed to SLN/CnEO (50 and 100 \(\mu\)L/L), and cell viability was evaluated by the MTT assay after 24 h. Results are expressed as the mean \(\pm\) SD (n = 6). Of the viability, 70% is indicated by the dotted line; below that, the treatment could be considered cytotoxic. free and encapsulated CnEO (Figure S2). It was observed that free CnEO did not increase cell death at the tested concentrations (50 and 100 \(\upmu\)L/L CnEO) after 24 h of incubation. In contrast, CnEO encapsulation strongly enhanced A549 cell death from 1.3 and 3.4% to 12.6 and 15.9% at 50 and 100 \(\upmu\)L/L CnEO, respectively (\(p<0.001\)). On the other hand, the mitochondrial membrane potential of A549 cells exposed to CnEO and SLN/CnEO was not altered at concentrations up to 200 \(\upmu\)L/L CnEO, neither at short (3 h) nor long (24 h) time incubations, suggesting that other mechanisms apart from MMP depolarization may be involved in A549 cell death, such as death-ligand-induced apoptosis, autophagy, or necroptosis [48; 49; 50; 51]. Due to the highly heterogeneous composition of EO as well as the wide variety of cancer types, defining an exclusive mechanism of action becomes challenging. Initially, research focused on exploring the antioxidant and anti-inflammatory properties of EO, suggesting their potential for treating cancer. Cancer cells are characterized by a moderate increase in reactive oxygen species (ROS) compared to normal cells. Moreover, inflammation is also considered a hallmark of cancer [4; 50; 51]. On the other hand, EO, or their main VOC, may also exhibit anticancer effects by increasing ROS generation [26; 47; 51]. It is well established that ROS modulate signaling pathways involved in cell survival and proliferation, such as MAP kinases and Akt/mTOR, as well as EO and VOC, and are capable of directly interfering with these pathways, leading to cancer cell death [47; 50; 51]. In a previous study, we found that CnEO and _L. alba_ (chemotype linalool) essential oils did not present significant antioxidant capacity but, in contrast, demonstrated pro-oxidant activity on low-density lipoproteins (LDL) [22]. Here, we found that CnEO encapsulation enhanced cell viability inhibition and promoted cell death independently of MMP collapse; however, further studies are required to shed light on the antiproliferative mechanisms exerted by SLN/CnEO. Cell migration, a feature of tumor spread and cancer invasion, is a key aspect to consider. Indeed, lung cancer is among the malignancies that most commonly metastasize [47].