Cytotoxic Screening and Enhanced Anticancer Activity of Lippia alba and Clinopodium nepeta Essential Oils-Loaded Biocompatible Lipid Nanoparticles against Lung and Colon Cancer Cells

Plant and herbal essential oils (EOs) offer a wide range of pharmacological actions that include anticancer effects. Here, we evaluated the cytotoxic activity of EO from Lippia alba (chemotype linalool), L. alba (chemotype dihydrocarvone, LaDEO), Clinopodium nepeta (L.) Kuntze (CnEO), Eucalyptus globulus, Origanum × paniculatum, Mentha × piperita, Mentha arvensis L., and Rosmarinus officinalis L. against human lung (A549) and colon (HCT-116) cancer cells. The cells were treated with increasing EO concentrations (0–500 µL/L) for 24 h, and cytotoxic activity was assessed. LaDEO and CnEO were the most potent EOs evaluated (IC50 range, 145–275 µL/L). The gas chromatography–mass spectrometry method was used to determine their composition. Considering EO limitations as therapeutic agents (poor water solubility, volatilization, and oxidation), we evaluated whether LaDEO and CnEO encapsulation into solid lipid nanoparticles (SLN/EO) enhanced their anticancer activity. Highly stable spherical SLN/LaDEO and SLN/CnEO SLN/EO were obtained, with a mean diameter of 140–150 nm, narrow size dispersion, and Z potential around −5mV. EO encapsulation strongly increased their anticancer activity, particularly in A549 cells exposed to SLN/CnEO (IC50 = 66 µL/L CnEO). The physicochemical characterization, biosafety, and anticancer mechanisms of SLN/CnEO were also evaluated in A549 cells. SLN/CnEO containing 97 ± 1% CnEO was highly stable for up to 6 months. An increased in vitro CnEO release from SLN at an acidic pH (endolysosomal compartment) was observed. SLN/CnEO proved to be safe against blood components and non-toxic for normal WI-38 cells at therapeutic concentrations. SLN/CnEO substantially enhanced A549 cell death and cell migration inhibition compared with free CnEO.


Cell Culture
Human colorectal carcinoma HCT-116 (CCL-247) and lung adenocarcinoma A549 (CCL-185) cells were provided by the American Type Culture Collection (ATCC). WI-38 cells (normal human embryonic lung fibroblasts, ATCC CCL-75) were a kind gift from Dr. Natalia Scaglia (School of Medicine, National University of La Plata). Cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% P/S (Gibco) at 37 • C under a 5% CO 2 atmosphere.

Cytotoxic Screening of Different EOs
The cytotoxic activity of EO was determined by the MTT assay [23]. HCT-116 (5 × 10 3 ) and A549 (5 × 10 3 ) cells were placed in a 96-well microplate and kept for 24 h at 37 • C and 5% CO 2 . Afterward, the medium was discarded and replaced with increasing concentrations (0-500 µL/L) of the eight mentioned EO dissolved in 100% ethanol (final concentration < 0.1%) in 10% FBS-supplemented DMEM for 24 h. After rinsing with PBS, cells were incubated with MTT solution (0.5 mg/mL in serum-free DMEM) for 3 h. The produced formazan was dissolved in 100 µL DMSO, the plates were shaken for 10 min, and the absorbance at λ = 560 nm was measured in a microplate reader (Beckman Coulter DTX 880, USA). Cell viability was expressed as a percentage of the untreated control (100% survival).

Analysis by Gas Chromatography-Mass Spectrometry
The volatile organic compound (VOC) composition of EO was analyzed with a Hewlett-Packard 6890 gas chromatograph (GC) coupled to a mass-selective detector (Agilent 5975C VL) as previously described [22]. Briefly, VOC separation was performed employing a ZB-5HT Inferno column (30 m, 0.25 mm d.i., 0.25 µm film, Phenomenex, Torrance, CA, USA). The injector was operated at 250 • C. The oven was programmed as follows: 40 • C for 1 min; 10 • C/min to 300 • C with a holding time of 3 min at the final temperature. The MSD was set at 70 eV and operated in scan mode with a mass range of 35-600 amu; transference line at 300 • C; ionization chamber at 250 • C; and quadrupole at 150 • C. The samples were obtained by head space-solid phase microextraction (HS-SPME). A preconditioned 65 µm polydimethylsiloxane/divinylbenzene fiber (PDMS/DVB, Supelco, Bellefonte, PA, USA) was exposed for 2 min to 1 µL of each EO placed in a 2 mL glass vial sealed with a Teflon cover with a rubber seal at 25 • C. VOC were identified by interpretation of their mass fragmentation pattern; spectra were also compared to data from MS libraries (NIST 05 Mass Spectral Library) and the literature (Adams, 2007). VOC chain lengths were confirmed by calculating their Kovats index (KI).

Preparation of EO-Loaded SLN (SLN/EO)
The SLN/EO were prepared through homogenization by the ultrasonication method, as previously reported by our group [24]. In a pre-warmed water bath at 70 • C, 200 mg MM (2.0% w/v) was first melted, and then 250 µL EO was added. Ten milliliters of a pre-warmed aqueous solution of poloxamer 188 (3.0% wt) were added to the lipidic phase. Rapidly, the pre-emulsion was subjected to ultrasonication for 10 min (40% amplitude) in a Cole-Parmer ultrasonic processor (130 W, USA). For control experiments, empty SLNs were prepared following the same procedure but omitting the addition of EO.

Transmission Electron Microscopy (TEM)
The TEM analysis was carried out in a Jeol-1200 EX II-TEM microscope (Jeol, MA, USA) as described earlier [20].

Particle Size, Zeta Potential, and Polydispersity Index
Nanoparticle mean diameter, size distribution (polydispersity index, PDI), and zeta potential (Z-pot) were measured in a Nano ZS Zetasizer Instrument (Malvern Instruments Corp., UK) at 25 • C, as previously described [25].

Cytotoxic Activity of Selected Free and Encapsulated EO
HCT-116 (5 × 10 3 ) and A549 (5 × 10 3 ) cells were plated in 96-well plates for 24 h under standard conditions and treated with increasing concentrations (25-400 µL/L EO) of free and/or encapsulated EO for 24 h. Cell viability was determined by MTT, as described earlier. This range (25-400 µL/L EO) was chosen based on previous results to cover nontoxic (25 µL/L) to highly cytotoxic (400 µL/L EO) concentrations of LaDEO and CnEO in both HCT-116 and A549 cells, therefore evidencing the potential enhancement of anticancer activity after encapsulation.

Encapsulation Efficiency (EE)
Percent EE for SLN/CnEO was determined indirectly by calculating the amount of non-encapsulated CnEO [20] by UV-Vis spectroscopy (λ max = 258 nm). Percent EE was calculated as: where Q 0 = initial quantity of CnEO; Cr = concentration of CnEO in the filtered solution; and V = total volume. The same procedure was followed for the empty SLN to discard possible interferences in UV-Vis detection.

Physical Stability
The SLNs were placed at 4 • C, protected from light, for six months, and their stability was followed by analyzing changes in mean particle size, PDI, and Z-pot.

Release Studies
CnEO release was determined as previously described [21] using two different buffer systems: phosphate buffer (pH 7.4) and acetate/acetic acid buffer (AC-+ AcH, pH 5.0). The CnEO concentration was measured at λ max = 258 nm using a UV-Vis spectrophotometer (Shimadzu, Japan).

Hemotoxicity Studies
Heparinized venous blood from healthy donors was used after obtaining the corresponding written informed consent. The blood was placed in a six-well plate in Ham F12 culture medium containing 10% FBS and exposed to increasing amounts of SLN, free-CnEO, and SLN/CnEO (100-400 µL/L CnEO) at 37 • C for 24 or 48 h. After centrifuging the mixture at 2500× g for 5 min, the precipitate was discarded. The proportion of lysed red blood cells was quantified by measuring the released hemoglobin at λ = 540 nm. Hemolysis (100%) was determined by exposing erythrocytes to 1.0% Triton X-100, while the negative control was obtained by incubating erythrocytes in phosphate-buffered saline (PBS).

Cytotoxic Activity of SLN/CnEO on Normal Lung WI-38 Fibroblasts
WI-38 cells were plated in 96-well plates at a density of 8 × 10 3 cells/well for 24 h under standard conditions and treated with concentrations of SLN/CnEO (50 and 100 µL/L CnEO) that significantly inhibited A549 lung cancer cells. Cell viability was determined by MTT, as described earlier.

Inhibition of Cell Migration
A549 cells (7.5 × 10 4 ) were seeded in 24-well plates for 24 h. The monolayers were scratched with a sterile pipette tip (200 µL) on the midline of the well and washed with DMEM to remove the detached cells. Then, the cells were exposed to DMEM containing 0.1% ethanol (Control), CnEO (50 and 100 µL/L), or SLN/CnEO (50 and 100 µL/L CnEO). A549 cell migration was assessed employing a Fluorescence Microscope (Olympus LX71 Inverted Tokyo, Japan) at 0 and 48 h. The wound healing area was defined employing Image J software (1.53k, NIH, USA) [26].

Statistical Analysis
Experimental data are expressed as means ± standard deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA), the Tukey-Kramer multiplecomparison test (significance level set at p < 0.05), or the unpaired t-test. Cell viability Non-linear regression curves (SigmaPlot software 14.0; Systat Software, Inc., Point Richmond, CA, USA) were used to calculate the IC 50 values for cell viability.

Chemical Composition of EO
LaDEO and CnEO chemical compositions (compounds higher than 1.0%) were analyzed ( Table 1). The complete CnEO composition was previously reported by our group [22]. As expected, most of the compounds identified were monoterpenes. The major constituents of LaDEO were dihydrocarvone isomer 1 (29.6%), limonene (25.2%), and dihydrocarvone isomer 2 (23.8%). It has been reported that within the L. alba species, limonene is frequently present as one of the major compounds of EO, accompanied by at least one of the five monoterpenic ketones, including dihydrocarvone [27].  On the other hand, pulegone (37.2%), menthone (26.6%), and isomenthone (11.7%) were the three most abundant monoterpenes found in CnEO. Božovic et al. [28] reported that at least three types of CnEO can be recognized. The most abundant components consist of C-3 oxygenated p-menthanes such as pulegone, menthone, isomenthone, and piperitone. Among them, pulegone is the main variant and the major component associated preferentially with menthone and/or isomenthone [28].
Our findings on EO composition are in line with those expected for LaDEO and CnEO according to literature data.

Synthesis of SLN/EO
The lipid matrix for nanoparticle synthesis used was MM, considering the lipophilic nature of the main VOCs, LaDEO and CnEO. MM is appropriate for the development of stable colloidal nanoparticles, as shown by earlier studies from our group [24].
A stable emulsion of EO in SLN was produced. TEM pictures revealed the existence of uniform and spherical nanoparticles ( Figure 1). Compared to empty SLN, the incorporation of CnEO into the MM matrix appeared to result in smaller nanoparticles.
were the three most abundant monoterpenes found in CnEO. Božovic et al. [28] reported that at least three types of CnEO can be recognized. The most abundant components consist of C-3 oxygenated p-menthanes such as pulegone, menthone, isomenthone, and piperitone. Among them, pulegone is the main variant and the major component associated preferentially with menthone and/or isomenthone [28].
Our findings on EO composition are in line with those expected for LaDEO and CnEO according to literature data.

Synthesis of SLN/EO
The lipid matrix for nanoparticle synthesis used was MM, considering the lipophilic nature of the main VOCs, LaDEO and CnEO. MM is appropriate for the development of stable colloidal nanoparticles, as shown by earlier studies from our group [24].
A stable emulsion of EO in SLN was produced. TEM pictures revealed the existence of uniform and spherical nanoparticles ( Figure 1). Compared to empty SLN, the incorporation of CnEO into the MM matrix appeared to result in smaller nanoparticles. The outcomes of the TEM images were confirmed by a dynamic light scattering (DLS) analysis ( Figure 2). For nanoparticles dispersed in aqueous environments, DLS is a more suitable and less-invasive technique than TEM since it reflects the presence of agglomerates and aggregates in a more representative way (indicated by the PDI parameter). The mean diameters of SLN, SLN/LaDEO, and SLN/CnEO were 147, 151, and 141 nm, respectively. The PDI was ≤0.3 in all cases, indicating high compatibility for biomedical purposes [29]. Indeed, SLN/LaDEO and SLN/CnEO displayed a PDI ≤ 0.2, whereas SLN presented a PDI of 0.24. These findings may be attributed to the good emulsion dispersion but also suggest that EO inclusion would promote a reorganization in the SLN structure [20,21,24]. This observation became even clearer when a population of SLN particles larger than 1000 nm vanished after being combined with EO ( Figures 1 and 2A), highlighting the monodispersed nature of the SLN/EO formulations. A decrease in the Z-pot of SLN from −12 mV to −5/6 mV was found after the incorporation of EO, which further reinforces the idea that EO would be crucial for SLN restructuring. The outcomes of the TEM images were confirmed by a dynamic light scattering (DLS) analysis ( Figure 2). For nanoparticles dispersed in aqueous environments, DLS is a more suitable and less-invasive technique than TEM since it reflects the presence of agglomerates and aggregates in a more representative way (indicated by the PDI parameter). The mean diameters of SLN, SLN/LaDEO, and SLN/CnEO were 147, 151, and 141 nm, respectively. The PDI was ≤0.3 in all cases, indicating high compatibility for biomedical purposes [29]. Indeed, SLN/LaDEO and SLN/CnEO displayed a PDI ≤ 0.2, whereas SLN presented a PDI of 0.24. These findings may be attributed to the good emulsion dispersion but also suggest that EO inclusion would promote a reorganization in the SLN structure [20,21,24]. This observation became even clearer when a population of SLN particles larger than 1000 nm vanished after being combined with EO ( Figures 1 and 2A), highlighting the monodispersed nature of the SLN/EO formulations. A decrease in the Z-pot of SLN from −12 mV to −5/6 mV was found after the incorporation of EO, which further reinforces the idea that EO would be crucial for SLN restructuring.

Cytotoxic Activity of Free and Encapsulated LaDEO and CnEO
As shown in Section 3.1, LaDEO and CnEO were the most active EOs against lung A549 and colon HCT-116 cells. Nevertheless, EO employment in pharmaceutical products has some restrictions due to its high volatility, low aqueous solubility, chemical instability, and low bioavailability [30]. Intending to determine the potential advantages of EO encapsulation into SLN, the cytotoxic activity of free and SLN/EO (SLN/LaDEO and SLN/CnEO) was explored in A549 and HCT-116 cells. The cells were incubated with increasing amounts of free and encapsulated EO (25- 100 µL/L, respectively (p < 0.001). Empty SLN in equivalent quantities did not produce any cytotoxicity at all in any of the two cell lines ( Figure 3C) [31].

Cytotoxic Activity of Free and Encapsulated LaDEO and CnEO
As shown in Section 3.1, LaDEO and CnEO were the most active EOs against lung A549 and colon HCT-116 cells. Nevertheless, EO employment in pharmaceutical products has some restrictions due to its high volatility, low aqueous solubility, chemical instability, and low bioavailability [30]. Intending to determine the potential advantages of EO encapsulation into SLN, the cytotoxic activity of free and SLN/EO (SLN/LaDEO and SLN/CnEO) was explored in A549 and HCT-116 cells. The cells were incubated with increasing amounts of free and encapsulated EO (25- Figure 3A; p < 0.001 in both cases). The effect of EO nanoencapsulation against A549 cells was most important since a significantly enhanced cytotoxic activity was observed from 50 µL/L CnEO and 100 µL/L LaDEO ( Figure  3B). A549 cell viability decreased from 96.5 to 63.2% and from 64.6 to 31.3% after encapsulation of 100 µL/L and 200 µL/L LaDEO, respectively (p < 0.001). CnEO incorporation into SLN decreased cell viability from 103.4 to 60.6% at 50 µL/L, and from 68.8 to 35.1% at 100 µL/L, respectively (p < 0.001). Empty SLN in equivalent quantities did not produce any cytotoxicity at all in any of the two cell lines ( Figure 3C) [31].
The improvement of EO activity by encapsulation may be attributed to a series of aspects such as extended EO stability in the culture medium at 37 °C, augmented EO permeation through the cell membrane, the sustained presence of cytotoxic amounts of EO resulting from a controlled release from SLN, possible evasion of expulsive mechanisms, and rapid metabolism of the main bioactive VOC due to SLN protection [32][33][34]. The improvement of EO activity by encapsulation may be attributed to a series of aspects such as extended EO stability in the culture medium at 37 • C, augmented EO permeation through the cell membrane, the sustained presence of cytotoxic amounts of EO resulting from a controlled release from SLN, possible evasion of expulsive mechanisms, and rapid metabolism of the main bioactive VOC due to SLN protection [32][33][34].
The IC 50 values of SLN/LaDEO and SLN/CnEO in A549 and HCT-116 cells are shown in Table 2. Based on these findings, the most promising system (SLN/CnEO against A549 cells) was explored for further studies.

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 λ 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].

SLN/CnEO Biocompatibility
One of the primary objectives of cancer therapy is to kill tumor cells by preventing, or at least minimizing, detrimental side effects on healthy organs.
The SLN/CnEO hemotoxicity was tested as a first step in identifying the potential negative effects of the formulation ( Figure 5A,B). The interaction with erythrocytes becomes an important factor when assessing the safety of nanoparticles, particularly if intravenous delivery is thought of as an SLN potential route [41]. According to the ISO/TR 7406 standard, biomaterials that result in a hemolytic ratio of less than 5% can be deemed safe for biomedical uses [42]. Here, erythrocytes were exposed to very high concentrations (100-400 µL/L) of free or encapsulated CnEO and long exposure periods (up to 48 h) to identify the safety of the formulations. A dose-dependent hemotoxicity of free CnEO was observed, which was significant at the highest tested concentration (400 µL/L) ( Figure  5A,B). On the other hand, hemolytic ratios of less than 3.5% were found at any condition analyzed in SLN/CnEO. Probably, SLN may be protecting erythrocytes from direct CnEO exposure, where the main VOCs could be destabilizing their membranes, leading to hemolysis.
In addition, the cytotoxicity of SLN/CnEO on WI-38 normal lung fibroblasts was examined. Figure 5C shows that SLN/CnEO was non-toxic for WI-38 cells (viability > 70%) at 50 and 100 µL/L CnEO, which inhibited about 40 and 65% A549 cell viability, respectively ( Figure 3B). This specific activity dependent on the cell type (normal or tumoral) may be associated with the selective activity of monoterpenes against cancer cells previously described [26,43], although further studies are required to shed light on this issue.
Altogether, these findings suggest that SLN/CnEO is a biocompatible and safe system able to improve the anticancer activity of CnEO. 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 • 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.

SLN/CnEO Biocompatibility
One of the primary objectives of cancer therapy is to kill tumor cells by preventing, or at least minimizing, detrimental side effects on healthy organs.
The SLN/CnEO hemotoxicity was tested as a first step in identifying the potential negative effects of the formulation (Figure 5A,B). The interaction with erythrocytes becomes an important factor when assessing the safety of nanoparticles, particularly if intravenous delivery is thought of as an SLN potential route [41]. According to the ISO/TR 7406 standard, biomaterials that result in a hemolytic ratio of less than 5% can be deemed safe for biomedical uses [42]. Here, erythrocytes were exposed to very high concentrations (100-400 µL/L) of free or encapsulated CnEO and long exposure periods (up to 48 h) to identify the safety of the formulations. A dose-dependent hemotoxicity of free CnEO was observed, which was significant at the highest tested concentration (400 µL/L) ( Figure 5A,B). On the other hand, hemolytic ratios of less than 3.5% were found at any condition analyzed in SLN/CnEO. Probably, SLN may be protecting erythrocytes from direct CnEO exposure, where the main VOCs could be destabilizing their membranes, leading to hemolysis. Of the viability, 70% is indicated by the dotted line; below that, the treatment could be considered cytotoxic.

Anticancer Mechanisms of CnEO and SLN/CnEO
Essential oils and their major VOCs, monoterpenes, 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 monoterpene-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 free and encapsulated CnEO ( Figure S2). It was observed that free CnEO did not increase cell death at the tested concentrations (50 and 100 µL/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 µL/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 µL/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]. In addition, the cytotoxicity of SLN/CnEO on WI-38 normal lung fibroblasts was examined. Figure 5C shows that SLN/CnEO was non-toxic for WI-38 cells (viability > 70%) at 50 and 100 µL/L CnEO, which inhibited about 40 and 65% A549 cell viability, respectively ( Figure 3B). This specific activity dependent on the cell type (normal or tumoral) may be associated with the selective activity of monoterpenes against cancer cells previously described [26,43], although further studies are required to shed light on this issue.
Altogether, these findings suggest that SLN/CnEO is a biocompatible and safe system able to improve the anticancer activity of CnEO.

Anticancer Mechanisms of CnEO and SLN/CnEO
Essential oils and their major VOCs, monoterpenes, 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 monoterpene-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 free and encapsulated CnEO ( Figure S2). It was observed that free CnEO did not increase cell death at the tested concentrations (50 and 100 µL/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 µL/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 µL/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]. Bioactive compounds with cytotoxic and anti-metastatic properties are ideal candidates. Therefore, the impact on cell migration was investigated using the wound healing assay to better understand both the anti-cancer mechanisms of free CnEO and the advantages of its encapsulation in SLN ( Figure 6). We observed that 100 µL/L CnEO inhibited cell migration to some degree (p < 0.001), whereas SLN/CnEO impaired the migration of A549 cells at lower concentrations (50 µL/L) and significantly improved the anti-migratory effects of free-CnEO ( Figure 6B, p < 0.001 vs. control cells and free-CnEO), suggesting that CnEO had antimetastatic effects on lung A549 cells, which are definitely exacerbated after its encapsulation into SLN.

Conclusions
Our findings revealed that LaDEO and CnEO were the most active among eight different EOs against both lung cancer (A549) and colon cancer (HCT-116) cells. Encapsulation of LaDEO and CnEO into SLN improved the anticancer effects of free EO in both cell lines. SLN/CnEO in A549 cells resulted in the most effective system. A deeper characterization of SLN/CnEO revealed a high EE of CnEO and an interesting pH dependency of CnEO release from SLN, favoring CnEO release in acidic conditions, which mimicked the endolysosomal compartment. SLN/CnEO seemed to be highly biocompatible since no toxic effects on red blood cells or normal lung fibroblasts were observed at concentrations at which A549 cancer cells were completely killed. In contrast to free CnEO, SLN/CnEO induced significant A549 cell death that seemed to be independent of the mitochondrial depolarization pathway and substantially enhanced the inhibition of A549 cell migration mediated by free CnEO.
The obtained formulations could be considered green anticancer tools in adjuvant and/or complementary therapies. However, additional investigations focusing on molecular mechanisms and in vivo studies are recommended. This information may be useful

Conclusions
Our findings revealed that LaDEO and CnEO were the most active among eight different EOs against both lung cancer (A549) and colon cancer (HCT-116) cells. Encapsulation of LaDEO and CnEO into SLN improved the anticancer effects of free EO in both cell lines. SLN/CnEO in A549 cells resulted in the most effective system. A deeper characterization of SLN/CnEO revealed a high EE of CnEO and an interesting pH dependency of CnEO release from SLN, favoring CnEO release in acidic conditions, which mimicked the endolysosomal compartment. SLN/CnEO seemed to be highly biocompatible since no toxic effects on red blood cells or normal lung fibroblasts were observed at concentrations at which A549 cancer cells were completely killed. In contrast to free CnEO, SLN/CnEO induced significant A549 cell death that seemed to be independent of the mitochondrial depolarization pathway and substantially enhanced the inhibition of A549 cell migration mediated by free CnEO.
The obtained formulations could be considered green anticancer tools in adjuvant and/or complementary therapies. However, additional investigations focusing on molecular mechanisms and in vivo studies are recommended. This information may be useful for the development of innovative treatment modalities, including the incorporation of conventional lipophilic chemotherapeutics into SLN/EO.