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

Abstract

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.

Keywords: anticancer mechanisms; biocompatibility; cancer cells; drug delivery; essential oils; solid lipid nanoparticles.

Figure 1

TEM images of SLN (A), SLN/LaDEO (B), and SLN/CnEO (C). Scale bar: 200 nm.

Figure 2

DLS analysis. (A) Particle size distribution, (B) Polydispersity index (PDI), and (C) Z-potential (Z-pot). The results express the mean ± 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 ± 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).

Figure 4

CnEO EE and cumulative release. (A) CnEO EE (%). (B) CnEO release from SLN/CnEO at pH 7.4 and 5.0. Values are expressed as the mean ± SD (n = 3).

Figure 5

Biocompatibility of SLN/CnEO. (A,B) Hemotoxicity of empty SLN, free CnEO, and SLN/CnEO. Hemotoxicity of 100, 200, and 400 µL/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 ± 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 µL/L), and cell viability was evaluated by the MTT assay after 24 h. Results are expressed as the mean ± SD (n = 6). Of the viability, 70% is indicated by the dotted line; below that, the treatment could be considered cytotoxic.

Figure 6

Encapsulation of CnEO increases the inhibition of A549 cell migration. Cell migration was analyzed by the wound healing assay. Cells were exposed to 0.1% ethanol (control), empty SLN, free CnEO, or SLN/CnEO (50 and 100 µL/L) for 48 h. (A) Representative images were obtained at 0 and 48 h (40×). (B) Quantitative analysis of wound healing closure. Data are presented as the mean ± SD (n = 4). (a) p < 0.05 vs. Control; (b) p < 0.05 vs. equivalent concentration of free CnEO.