Co-Delivery of Ylang Ylang Oil of Cananga odorata and Oxaliplatin Using Intelligent pH-Sensitive Lipid-Based Nanovesicles for the Effective Treatment of Triple-Negative Breast Cancer

Abstract

Smart pH-responsive niosomes loaded with either Oxaliplatin (Ox), Ylang ylang essential oil (Y-oil), or co-loaded with both compounds (Ox-Y) (Ox@NSs, Y@NSs, and Ox-Y@NSs, respectively) were formulated utilizing the thin film method. The developed nanocontainers had a spherical morphology with mean particle sizes lower than 170 nm and showed negative surface charges, high entrapment efficiencies, and a pH-dependent release over 24 h. The prepared pH-responsive niosomes' cytotoxicity was tested against the invasive triple-negative breast cancer (MDA-MB-231) cells, compared to free OX and Y-oil. All niosomal formulations loaded with Ox and/or Y-oil significantly improved cytotoxic activity relative to their free counterparts. The Ox-Y@NSs demonstrated the lowest IC50 (0.0002 µg/mL) when compared to Ox@NSs (0.006 µg/mL) and Y@NSs (18.39 µg/mL) or unloaded Ox (0.05 µg/mL) and Y-oil (29.01 µg/mL). In addition, the percentages of the MDA-MB-231 cell population in the late apoptotic and necrotic quartiles were profoundly higher in cells treated with the smart Ox-Y@NSs (8.38% and 5.06%) than those exposed to free Ox (7.33% and 1.93%) or Y-oil (2.3% and 2.13%) treatments. Gene expression analysis and protein assays were performed to provide extra elucidation regarding the molecular mechanism by which the prepared pH-sensitive niosomes induce apoptosis. Ox-Y@NSs significantly induced the gene expression of the apoptotic markers Tp53, Bax, and Caspase-7, while downregulating the antiapoptotic Bcl2. As such, Ox-Y@NSs are shown to activate the intrinsic pathway of apoptosis. Moreover, the protein assay ascertained the apoptotic effects of Ox-Y@NSs, generating a 4-fold increase in the relative protein quantity of the late apoptotic marker Caspase-7. Our findings suggest that combining natural essential oil with synthetic platinum-based drugs in pH-responsive nanovesicles is a promising approach to breast cancer therapy.

Keywords: apoptosis; cytotoxicity; essential oils; intrinsic pathway; invasive breast cancer; niosomes; oxaliplatin; platinum-based anticancer drugs.

Figure 1

GC–MS representative chromatogram of Y-oil (Cananga Odorata Hook. F. and Thomson).

Figure 2

TEM images of (A) blank NSs, (B) Y@NSs, (C) Ox@NSs, and (D) Ox-Y@NSs, demonstrating the spherical morphology of the prepared pH-responsive niosomes.

Figure 3

Release [%] of Y-oil, Ox, and Ox-Y from pH-responsive Y@NSs, Ox@NSs, and Ox-Y@NSs into phosphate buffer (pH 7.4) and acetate buffer (pH 5.5.4). Data are presented as mean ± SD; n = 3.

Figure 4

The cell viability of MDA-MB-231 cells after exposure to the different agents for 72 h. Data are displayed as the mean of three individual runs ± SD. The symbol (*) refers to the statistical significance of the Blank NSs-treated cells (Control). The symbol (♦) refers to the statistical significance of Y-oil treated cells. The symbol (●) refers to the statistical significance of Ox-treated cells. The symbol (■) refers to the statistical significance of Ox@NSs. The presence of any symbol once only indicates significance with a p-value ≤ 0.05. Any symbol repeated twice indicates significance with a p-value ≤ 0.01, and a symbol repeated thrice indicates a p-value ≤ 0.001.

Figure 5

A display of the apoptosis assay results in MDA-MB-231 cells when incubated for two consecutive days with the tested compounds (Ox, Y-Oil, and Ox-Y@NSs). Cytograms presenting annexin-V/Propidium Iodide-stained untreated MDA-MB231 cells as negative control (A), cells treated with Ox (B), cells treated with Y-Oil (C), cells treated with Ox-Y@NSs, (D) and analysis of the four quartiles resulting from the apoptosis assay (E). Quadrant charts show Q2-1 (necrotic cells, AV-/PI+), Q2-2 (late apoptotic cells, AV + /PI+), Q2-3 (normal cells, AV-/PI-) and Q2-4 (early apoptotic cells, AV + /PI-). Data are the average of three individual experiments ± standard deviation (SD). ***, ** and * refer to significant differences from the control, where p-values ≤ 0.001, ≤0.01 and ≤0.05, respectively.

Figure 6

The relative normalized gene expression of Bax (A), Bcl2 (B), Caspase-7 (C), and TP53 (D) after the exposure of MDA-MB-231cells to the test compounds (Y, Ox, and Ox-Y@NSs). Each value represents three replicates. β-actin gene was selected as the housekeeping gene and was implemented in data normalization in the RT-qPCR assay. The relative normalized gene expression was calculated using the 2^−ΔΔCt method. ***, ** and * refer to significant differences from the control, where p-values ≤ 0.001, ≤0.01 and ≤0.05, respectively.

Figure 7

Representative western blot for MDA-MB-231 cellular levels of Caspase-7 in the different treatment groups (A). Bar chart representation of the normalized Caspase-7 protein (B). Data are normalized using ß-actin and calculated relative to the control (untreated group). Data are presented as mean ± SD (n = 3). The symbol *** indicates a significant difference from untreated cells (control) at p < 0.001, respectively.