Anticancer Activity of Thymoquinone Cubic Phase Nanoparticles Against Human Breast Cancer: Formulation, Cytotoxicity and Subcellular Localization

Abstract

Introduction: Triple negative breast cancer is an aggressive disorder which accounts for at least 15% of breast cancer diagnosis and a high percentage of breast cancer morbidity, hence intensive research efforts are focused on the development of effective therapies to overcome the disease. Thymoquinone (TQ), the bioactive constituent of Nigella sativa, exhibits anticancer activity, yet its translation to the clinic is hindered by its poor bioavailability and lack of quantification method in blood and tissues. To overcome these limitations, cubosomes were utilized for the encapsulation and delivery of this anticancer molecule.

Methods: Thymoquinone loaded cubosomes were prepared through the emulsification homogenization method. The physicochemical characteristics, including particle size, zeta potential, morphology and entrapment efficiency, were studied. Moreover, the in vitro antitumor activity was tested on breast cancer cell lines (MCF-7 and MDA-MB-231) and compared to non-tumorigenic cell line (MCF-10A). Subcellular localization, cellular uptake and apoptotic effects of the formulations were assessed.

Results: The results revealed that the TQ loaded cubosomal formulation exhibited a mean particle size of 98.0 ± 4.10 nm with narrow unimodal distribution. The high entrapment efficiency (96.60 ± 3.58%) and zeta potential (31.50 ±4.20 mV) conceived the effectiveness of this nanosystem for TQ encapsulation. Cell viability in both breast cancer cell lines demonstrated a dose-dependent decrease in response to treatment with free TQ or TQ-loaded cubosomes, with enhanced antitumor activity upon treating with the latter formulation. A significant increase in apoptotic bodies and cleaved caspase 3 was observed upon treatment of MDA-MB-231 cells with either TQ or TQ-loaded cubosomes. Localization and trafficking studies unveiled that cubosomes accumulate in the cytoplasm of the studied breast cancer cell lines.

Discussion: Our results show that thymoquinone encapsulation in cubosomal nanoparticles provides a promising anticancer drug delivery system with the ability to label, detect and subsequently trace it within the human cells.

Keywords: antitumor; breast cancer; cubosomes; endocytosis; nanoparticles; thymoquinone; uptake.

Figure 1

Transmission electron microscope imaging of thymoquinone-loaded cubosomes.

Figure 2

Hemolytic activity and hemolysis percentage of TQ-loaded cubosomes (A) photograph of erythrocytes treated with TQ-loaded cubosomes at different percentage (v/v), (B) photomicrograph of erythrocytes treated with PBS (negative control), TQ-loaded cubosomes, Triton X (positive control), (C) Data expressed as mean ± SD of erythrocytes treated with different percentages of TQ-loaded cubosomes.

Figure 3

Toxicity of TQ-loaded cubosomes to non-tumorigenic breast cells and their anticancer effect in human breast cancer cells in comparison to free TQ. MTT assay showing the viability of (A) MCF-10A normal breast cell line. (B) MCF-7 breast cancer cell line. (C) MDA-MB-231 aggressive breast cancer cell line. The cells were treated for 24 h with different concentrations of either TQ, or blank cubosomes or TQ-loaded cubosomes. Experiments were repeated three times, data are means ± SEM, asterisk indicates p<0.05 with respect to the untreated control, bar and asterisk indicates p<0.05 of TQ-loaded cubosomes with respect to TQ, *p<0.05, **p<0.01, ***p<0.001.

Figure 4

(A) Immunofluorescent analysis of active caspase 3 (AC3) expression in MDA-MB-231 cell line after 24 h of treatment with TQ and TQ-loaded cubosomes using the IC₅₀ values obtained from MTT. Visualized by microscope Zeiss Axio, 40X oil immersion. (B) Active caspase 3 quantification in MDA-MB-231 cell line. Experiments were repeated three times, data are means ± SEM, asterisk indicates p<0.05 with respect to the untreated control, bar and asterisk indicates p<0.05 of TQ-loaded cubosomes with respect to TQ, *p<0.05, **p<0.01, ***p<0.001. Visualized by microscope Zeiss Axio, 40X oil immersion. Arrows indicate apoptotic bodies in the nuclei stained by DAPI.

Figure 5

Mechanism of cellular uptake and trafficking of TQ-loaded cubosomes in human breast cancer cell lines. (A) Cellular uptake of the TQ and blank formulations in MCF-7 and MDA-MB-231 cell lines after 30 mins of treatment with the IC₅₀ concentrations. (B) Subcellular localization of TQ-loaded cubosomes in MCF-7 and MDA-MB-231 cell lines. Cells were treated with 27.60 µM (MCF-7) and 7.60 µM (MDA-MB-231) of TQ-loaded cubosomes for 30 mins. The cubosomal formulations were labeled with nile red for tracing inside the cells. Slides were labeled with DAPI, LAMP, Caveolin, EEA-1 and Transferrin. Visualized by microscope Zeiss Axio, 40X oil immersion. Scale is 50 µm.