Chemical Investigation and Screening of Anti-Proliferative Activity on Human Cell Lines of Pure and Nano-Formulated Lavandin Essential Oil

Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding L. angustifolia × L. latifolia, is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.


Introduction
Plants provide a large amount of secondary metabolites, some of which are used in their original or semisynthetic form, to treat different human pathologies [1]. Among the secondary metabolites, essential oils, obtained by classical or by more modern methods of extraction from different parts of aromatic plants such as flowers, leaves, rhizomes, seeds, wood, fruits and bark [2,3], are a complex The antiproliferative activity of LEO on several human tumor cell lines including human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A), was investigated. The chemical composition and the activity exerted by the oil formulated in nanoemulsion (NanoLEO) on the same cell lines were also investigated to compare the biological effects with pure LEO. For this purpose, scanning and transmission electron microscopy investigations (SEM and TEM) were also carried out to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.

Nanoemulsion Characterization
LEO was formulated in nanoemulsion using a solvent displacement-evaporation technique, which did not require a high-energy input, as emulsification occurred spontaneously when the organic phase containing LEO was poured at room temperature into the aqueous phase. After evaporation of the organic solvents used, a nanometer-sized o/w emulsion with a narrow size distribution was formed. The formulation displayed a mean hydrodynamic diameter of 302.0 ± 2.4 nm, a PdI of 0.128 ± 0.001 and a ζ potential of +47.2 ± 1.6 mV, as determined by DLS and ELS (Dynamic Light Scattering and Electrophoretic Light Scatterin) respectively. The morphology, dimensions and homogeneity of NanoLEO were also evaluated by negative stain TEM, which evidenced the formation of spherical globules and confirmed the results from the DLS analysis.

Cytotoxicity Test (MTT)
To evaluate the cytotoxic effects of pure LEO and NanoLEO, a cell viability assay (MTT) was carried out on five different cell lines. The assay was performed both in a dose dependent and time dependent manner.
The effects of both LEO and NanoLEO were different between the cell lines considered ( Figure  2). In Figure 2a the EC50 values obtained after three different incubation times with LEO were represented and the Caco-2, the MCF7 and the MCF10A cells were more resistant to the treatment

Nanoemulsion Characterization
LEO was formulated in nanoemulsion using a solvent displacement-evaporation technique, which did not require a high-energy input, as emulsification occurred spontaneously when the organic phase containing LEO was poured at room temperature into the aqueous phase. After evaporation of the organic solvents used, a nanometer-sized o/w emulsion with a narrow size distribution was formed. The formulation displayed a mean hydrodynamic diameter of 302.0 ± 2.4 nm, a PdI of 0.128 ± 0.001 and a ζ potential of +47.2 ± 1.6 mV, as determined by DLS and ELS (Dynamic Light Scattering and Electrophoretic Light Scatterin) respectively. The morphology, dimensions and homogeneity of NanoLEO were also evaluated by negative stain TEM, which evidenced the formation of spherical globules and confirmed the results from the DLS analysis.

Cytotoxicity Test (MTT)
To evaluate the cytotoxic effects of pure LEO and NanoLEO, a cell viability assay (MTT) was carried out on five different cell lines. The assay was performed both in a dose dependent and time dependent manner.
The NanoLEO treatments demonstrated both a dose an time dependent effects: ranging from 0.57 × 10 −2 ± 0.08 × 10 −2 % for 24 h to 0.87 × 10 −2 ± 0.05 × 10 −2 % for 72 h for CCRF-CEM cells; from 0.64 × 10 −2 ± 0.05 × 10 −2 % for 24 h to 0.44 × 10 −2 ± 0.02 × 10 −2 % for 72 h for SHSY5Y; from 1.79 × 10 −2 ± 0.53 × 10 −2 % (24 h) to 1.58 × 10 −2 ± 0.09 × 10 −2 % for 72 h for Caco-2 cells; from 1.73 × 10 −2 ± 0.10 × 10 −2 % for 24 h to 1.35 × 10 −2 ± 0.21 × 10 −2 % for 72 h for the MCF7 cells; from 0.90 × 10 −2 ± 0.14 × 10 −2 % for 24 h to 0.25 × 10 −2 ± 0.14 × 10 −2 % for 72 h for the MCF10A cells (Table 2). The effects of both LEO and NanoLEO were different between the cell lines considered ( Figure 2). In Figure 2a the EC 50 values obtained after three different incubation times with LEO were represented and the Caco-2, the MCF7 and the MCF10A cells were more resistant to the treatment while the CCRF-CEM and the SHSY5Y cells resulted more sensitive. The EC 50 values obtained after the treatment with NanoLEO were reported in Figure 2b. The effects of LEO were slightly amplified in nanoformulation for CCRF-CEM and SHSY5Y; otherwise the MCF7, MCF10A and mostly CaCo-2 cells were even more affected by NanoLEO respect to LEO treatments. The DMSO control and the nanoemulsion control did not inhibit the proliferation of all the cell lines investigated. while the CCRF-CEM and the SHSY5Y cells resulted more sensitive. The EC50 values obtained after the treatment with NanoLEO were reported in Figure 2b. The effects of LEO were slightly amplified in nanoformulation for CCRF-CEM and SHSY5Y; otherwise the MCF7, MCF10A and mostly CaCo-2 cells were even more affected by NanoLEO respect to LEO treatments. The DMSO control and the nanoemulsion control did not inhibit the proliferation of all the cell lines investigated.

Statistical Analysis
The data from all independent experiments were expressed as means ± standard deviation (SD). Statistical analysis was performed using a one-way ANOVA test with a Stat-Plus software (AnalystSoft ©2009) with the threshold of significance set at p < 0.05.

Ultrastructure Investigation by Electron Microscopy
SEM and TEM investigations were carried out on Caco-2 cells exposed to LEO and NanoLEO treatments for 4 h. In Figure 3a  TEM analysis were also carried out to study the cell ultrastructure ( Figure 4). In control cells a typical cytoplasmic organization can be observed (Figure 4a), with a regular nucleus, endoplasmic reticulum, Golgi apparatus and numerous normal shaped mitochondria (Figure 4a, arrowhead). A higher magnification of mitochondrion is reported in Figure 4b.
In LEO treated cells (Figure 4c) some mitochondria showed modifications in their features. Mitochondria were swollen and with altered cristae structure (arrowhead) and a more electron dense matrix was observed. A higher magnification (Figure 4d

Discussion
Nowadays lavender essential oil is still investigated to better clarify its biological properties. The anticancer activities of essential oils from numerous plant species and their constituents have been studied in different human cell lines in vivo and in vitro as reviewed by Gautam et al. [38]. Once essential oils have penetrated into the cell membrane, they act through different mechanisms by which essential oils carry out their anticancer action including apoptosis, cell cycle arrest, antimetastatic and antiangiogenic, increased levels of reactive oxygen and nitrogen species, DNA repair modulation and others. Russo et al. [39], reported that different essential oils showed anticancer activities in vitro and in vivo models underlining among them those whose activities are due to the phytocomplex. Clear evidences were reported on apoptosis induction mechanism exerted by essential oils: Melissa officinalis essential oil induced apoptosis of glioblastoma multiforme cells [40], Salvia milthiorriza treatment reduced the proliferation of HepG2 hepatoma cells, changing their morphology and inducing cell death by apoptosis [41], Artemisia annua essential oil induced apoptosis in SMMC-7721 hepatocarcinoma cells [42] and essential oil of the conifer tree Tetraclinis articulata showed pro apoptotic activity in human melanoma and ovarian cancer cell lines [43]. In our previous paper, LEO treatment has been reported to induce apoptosis on HL60 human leukemia cells and, among the main LEO compounds, both terpinen-4-ol and linalyl acetate possess antiproliferative activity [28].
In the present paper, LEO and its nanoformulation NanoLEO have been analyzed by HS/GC-MS technique to describe the volatile chemical profile of their vapor phase. The results reported in Table 1 highlighted that linalool (LEO: 28.6% ± 0.04; NanoLEO: 60.4% ± 0.03) and 1,8-cineole (LEO: 27.4% ± 0.01; NanoLEO: 12.6% ± 0.01) were the most abundant components. Linalool showed cytotoxic activity on C32 cells line [44] and human leukemia and lymphoma cell lines [45]. In particular, linalool induced apoptosis of numerous leukemia cells by upregulation of p53 and cyclin-dependent kinase inhibitors [30]. Furthermore, other minor components in association, such as linalyl acetate, alpha-terpineol and camphor also found in LEO and NanoLEO, have been reported to be able to induce inhibition of the growth of the human colon cancer cell lines (HCT-116 p53+/+ and p53−/−) [46]. The cytotoxicity of 1,8-cineole was well described in more papers. For example, Hayes et al. [47] reported a moderate cytotoxic action on Hep G2, HeLa, MOLT-4, K-562 and CTVR-1 cell lines while Cha and colleagues [48] found that this compound, induced apoptosis of KB cells through caspase activation. LEO and NanoLEO have been then tested on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and on normal breast epithelial cells (MCF10A) in order to compare their biological activities. The screening of the antiproliferative activities for LEO and NanoLEO was carried out by MTT assay and the results revealed that the effects of LEO and NanoLEO were in a dose-and not time-dependent manner and different among the investigated cell lines (Figure 2). Concerning LEO, the EC 50 values showed that Caco-2, MCF7and MCF10A cells were more resistant to the treatment while the CCRF-CEM and the SHSY5Y cells were more sensitive. It was interesting to observe how the antiproliferative effect of LEO was amplified when the essential oil was supplied in nanoformulation (NanoLEO). Such differences were highly evident in the more resistant cell lines where the affection level was consistently higher after treatment with nanoformulated samples. Also, in the CCRF-CEM and SHSY5Y, the effects of LEO were amplified in nanoformulation although not so strongly.
Several studies on the effects of L. angustifolia extracts and the essential oil carried out on different cancer cell lines confirmed the observed cytotoxic properties of Lamiaceae EOs and their potential role as anticancer drugs. Tayarani-Najaran et al. [25] reported high antiproliferative activity of both L. angustifolia extract and essential oil and apoptotic mechanism induction in Hela and MCF-7 cells by EtOH and n-hexane extract. Furthermore, Nikoli'c et al. [21] found antiproliferative activity on Hela and A549 cells, with EC 50 values of 80.62 ± 1.04 µg/mL and 88.90 ± 1.71 µg/mL respectively. Gezici and collaborators [27] have also demonstrated the effects of lavender EOs at low concentrations and at a minimum exposure time. Moreover, studies on prostate cancer cells exhibited EC 50 values of 0.199% ± 0.026% for DU145 cells and 0.037% ± 0.011% (v/v) for PC-3 cells, confirming the potent cytotoxic activity of lavender EOs [34]. It has been reported that L. × intermedia showed cytotoxic effects on human breast cancer cells MCF-7 [49] and the cytomorphological modification could represent the activation of a programmed cell death. Studies on the effects of L. angustifolia extracts and the essential oil on Hela and MCF-7 reported of an antiproliferative action although an apoptotic mechanism induction was not demonstrated [25].
Nanotechnology has been shown to be a suitable strategy for cancer treatment and nanoemulsions might be the solution for different types of cancer (colon, ovarian, prostate, leukemia, breast, lung and melanoma). These formulations solve water-solubility problems, provide specific targeting to cancer cells and reduce tumor growth [50]. As reported in the studies on MCF 7 cells treated with Nigella stativa essential oil [51], nanoemulsions were tested to improve the effects of essential oils. In this case, a reduced cell viability and an alteration of nuclear morphology in a dose-and time-dependent manner was relieved. Zataria multiflora incapsulated essential oil was investigated in breast cancer cells and an enhanced in vitro anticancer activity with respect to the corresponding essential oil was observed [52]. Nanoemulsions based ginger and frankincense essential oils can ameliorate the apoptotic effects of mitomycin C on HeLa cervical cancer cells and MCF-7 breast cancer cells [53].
The employment of a hydrophobic core also allows the encapsulation of drugs. Liu et al. [54] reported that the quinolone alkaloid evodiamine formulated in nanoemulsion was capable of making sensitive cancerous A549 cells thanks to improved cellular uptake.
In our investigation, Caco-2 cell line cytotoxicity test showed a relevant difference in EC 50 values of LEO and NanoLEO treatments and the cancer colorectal cell line resulted much more sensitive to the nanoemulsion respects to not formulated LEO. In this view, Caco-2 cell line was further investigated by SEM and TEM to determine possible morphological alterations.
It has been reported that treatments with essential oils from four lavender species on Caco-2 cells determine an alteration of the cellular surface which become smooth with tiny microvilli [55]. Our SEM observations (Figure 3), showed alterations of the plasma membrane protrusions in LEO treated cells; the protrusions appeared shorter and not uniformly distributed. Few nanoemulsions particles were observed on the cell surfaces because of the 60 nm sections. The particles size, observed by the negative staining procedure, reveal a correspondence with the previous defined range size.
Due to the nanometer size, generally in the range of 10-500 nm, nanoemulsions, are able to enhance the activity of EOs since they allow a better and deeper tissue penetration and an easier cellular uptake. Moreover, they allow control and modulation of the release of active ingredients on the target site [36].
The cellular uptake of nanoemulsions may occur through different pathways, such as macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis and clathrin-and caveolae-independent endocytosis [56]. In particular, the mechanism of uptake of nanoemulsions has been reported to vary appreciably due to differences in emulsifiers, surface properties, particle shape and size [57]. About this last point, more studies reported that increased cellular uptake is linked to decreased droplet size [58,59]. Endocytosis of emulsions is a complex mechanism and more detailed information about cellular uptake and transport mechanism of lipid-based nanoemulsions are currently unavailable and further studies are required.
In the present study some evidences (Figure 3) showed morphological alterations in the cells after LEO and NanoLEO treatments. Mitochondrial morphology resulted to be deeply altered; mitochondria appeared to be swollen, with longitudinally rearranged or disorganized cristae and with more electron dense matrix mainly in NanoLEO rather than in LEO treatment. The loss of mitochondrial membrane integrity with the release of cytochrome-c and other proapoptotic factors in the cytosol and the remodeling of the cristae membrane is the first morphological alteration of the apoptotic process [60,61].
In NanoLEO treated cells and in minor extension in LEO treated cells, microtubule bundles were observed. Microtubule bundles, known as apoptotic microtubule networks, play particularly important functions in maintaining the plasma membrane integrity [62] and the dispersion of cellular and nuclear fragments [63] during execution apoptosis.
Taken together the morphological alterations particularly evident in NanoLEO treated cells, such as mitochondria modifications and the presence of apoptotic microtubules networks and the previous results of LEO treatment on HL60 cells [28], we can hypothesize that LEO and more efficiently NanoLEO treatments induce apoptotic cell death.
In conclusion, by the HS-GC/MS chemical analyses, we relieved a loss of some components and consequently a slight change in the volatile profile of the essential oil following its formulation in nanoemulsion. This change in the chemical composition, should lead to a reduction on the antiproliferative activity. On the contrary, we observed an enhanced effect on all the cell lines tested and in particular on the Caco-2 cell line. Therefore, considering the above, we can assume that differences evidenced in the behavior and activity of LEO, with respect to NanoLEO, may be attributed to the formulation which may have favored cellular uptake of essential oil.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Essential oil, pure and formulated, was analyzed by a gas chromatograph directly coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model (Waltham, MA, USA). The GC was equipped with a Restek Stabilwax (fused-silica) polar capillary column. Helium was used as carrier gas at a flow rate of 1 mL/min The injector was set to a 280 • C and the oven temperature program was as follows: isothermal at 60 • C for 5 min, then ramped to 220 • C at a rate of 5 • C min −1 and finally isothermal at 220 • C for 20 min. Front detector was an Electro Impact-Mass Spectrometer (EI-MS), mass spectra were recorded at 70 eV (EI) and were scanned in the range 40-400 m/z. Ion source and the connection parts temperature was 220 • C. The back detector was a FID (Flame Ionization Detector) maintained at 250 • C. The injector split ratio was 1:20. The GC-TIC (Total Ion Chromatogram) mass spectra were obtained by the TurboMass data analysis software (Perkin Elmer). The LEO constituents were identified by comparison of their linear retention indices (LRIs), (relative to C8-C30 aliphatic hydrocarbons, Ultrasci injected in the column at the same operating conditions described above) with the available retention data in the literature. Further identification of all components was made by matching their mass spectra with those stored in the Wiley and NIST 02 mass spectra libraries database. GC-FID analysis was performed under the same experimental conditions using the polar column as described for the GC-MS measurements. Relative percentages of all identified components, were obtained by peak area normalization from GC-FID chromatograms without the use of an internal standard or correction factors and expressed in percentages. All analyses were repeated twice.

Head Space GC-MS Analysis
The volatile composition of essential oil pure and formulated were performed by a Perkin Elmer Headspace Turbomatrix 40 (Waltham, MA, USA) autosampler connected to GC-MS. To optimize the headspace procedure for the determination of volatile organic compounds (VOCs), parameters such as equilibration time and temperature were optimized. The gas phase of the sealed vials was equilibrated for 20 min at 60 • C and was followed immediately by compound desorption into GC injector in splitless mode.

Nanoemulsion Preparation
The nanoemulsions were prepared following a procedure reported in Garzoli et al. [37]. The method followed for the nanoemulsion preparation was the solvent displacement technique, which involves the spontaneous emulsification of an organic phase added to an aqueous phase under magnetic stirring. More specifically, the organic phase consisted of 100 µL of LEO, 30 mg of SPC and 3 mg of benzalkonium chloride dissolved in a solvent mixture composed of 9.5 mL of acetone and 0.5 mL of ethanol. The addition of the organic phase to 10 mL of deionized water, maintained under magnetic stirring, led to the diffusion of the organic solvents and the immediate and spontaneous formation of the nanoemulsion, as evidenced by the milky appearance of the mixture. The organic solvents were removed by evaporation under reduced pressure until a final volume of 10 mL was achieved and the obtained formulation was labelled as Nano LEO. A control nanoemulsion was prepared with the same procedure but using Labrafac as the oily phase.

Physicochemical Characterization of Nanoemulsions
Hydrodynamic diameter (Z-average, nm), polydispersity index (PdI) and zeta potential (mV) were measured by Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) measurements with a Zetasizer Pro (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The DLS and ELS techniques used a photon correlator spectrometer equipped with a 4 mW He/Ne laser source operating at 633 nm. All measurements were performed in non-invasive back scattering (scattering angle of 173 • ) and were thermostatically controlled at 25 • C. The samples were appropriately diluted with demineralized water before analysis.
Ten 2-dilutions (0.1% to 1.95 × 10 −4 %) of essential oil were used and incubated for three different times. DMSO and a nanoemulsion prepared using Labrafac (a compound not having any cytotoxic effect as alone), as the oily phase and stabilized with benzalkonium chloride and SPC, at the 0.1% were used as control. Vinblastine was used as positive control (ten 2-dilutions from 0.25 µM to 0.48 × 10 −3 µM).
After 24, 48 and 72 h, the medium containing treatments was removed and 100 µL of MTT solution (0.5 mg/mL) was added to each well and incubated in the dark at 37 • C for 3 h. The formazan crystals were dissolved in 100 µL of DMSO and the absorbance measured at 570 nm. The concentration at which a substance exerts half of its maximal response values (EC 50 ) was calculated by using AAT Bioquest, Inc. (Sunnyvale, CA 94085, USA) (5 November 2019).

Electron Microscopy
For the electron microscopy analysis, 1 × 10 5 Caco-2 cells were seeded onto a 24-well plate inserts and incubated for 24 h in appropriate culture conditions before treatments with Leo and NanoLEO at EC 50 concentrations.
After 4 and 24 h, the cells were collected in tubes, washed with PBS and fixed with 4% paraformaldehyde and 5% glutaraldehyde, pH 7.2, in a 0.1 M cacodylate buffer for 1 h at 4 • C [64]. After rinsing overnight in the same buffer, samples were post-fixed in 1% osmium tetroxide in a cacodylate buffer for 1 h at 4 • C. After two washings in the same buffer, samples were dehydrated in a graded ethanol series.
For Scanning Electron Microscopy (SEM), cells were dried by the critical point method using CO 2 in a Balzers Union CPD 020, sputter-coated with gold in a Balzers MED 010 unit and observed by a JEOL JSM 5200 electron microscope (Jeol Ltd., Tokyo, Japan).
For Transmission Electron Microscopy (TEM), samples were fixed and dehydrated as described above and embedded in an Epon mixture resin. Thin sections (50-70 nm) were cut with Reichert Ultracut (Leica Microsystems, Wetzlar, Germany) and LKB Nova ultramicrotomes (LKB Vertriebs GmbH, Vienna, Austria) using a diamond knife, collected on copper grids, stained with uranyl acetate and lead citrate and observed by a JEOL 1200EX II electron microscope.
For negative staining, the droplets of sample suspensions (10 µL) were placed on formvar-carbon coated grids and allowed to adsorb for 60 s. Excess liquid was removed gently touching the filter paper. The adsorbed specimen was then processed for negative-staining, by first washing the specimen grid on a drop of negative stain (2% uranyl acetate in distilled water), blotting and repeating this step once more, this time leaving the specimen grid for 60 seconds on a new drop of negative stain solution. Samples were observed at a JEOL 1200 EX II electron microscope. Micrographs were acquired by the Olympus SIS VELETA CCD camera equipped the iTEM software.

Conclusions
In this study, the chemical volatile composition of Lavandula × intermedia essential oil pure and encapsulated in nanoemulsion was characterized by HS-GC/MS technique and their antiproliferative activity investigated on different cell lines. The effects of LEO and NanoLEO occurred in a dose-and not time-dependent manner and in some of the tested cell lines NanoLEO formulation showed higher antiproliferative activity respect to LEO. Electron microscopy observations showed mitochondria modifications and apoptotic microtubules networks in LEO and more efficiently NanoLEO treatments, probably due to an apoptotic process. The mechanism of permeation of LEO and NanoLEO into the cells has not yet been established and it could represent an area of future investigation.
Since, to our knowledge, no study on comparative antiproliferative activity of LEO vs. NanoLEO has been conducted so far, this work is the first contribution for a better knowledge of the effectiveness of lavandin formulated oil compared to the pure essential oil.