Anticancer activity and uptake mechanisms of thymoquinone-loaded cubosomal nanoparticles in human breast cancer

Abstract

Background: Thymoquinone (TQ), the bioactive constituent of Nigella sativa (Black seed), exhibits anticancer potential, but its translation to the clinic is hindered by its hydrophobic property and poor bioavailability. Nanoparticles (NP) are evolving as a therapeutic platform for cancer treatment. In this project, we encapsulated TQ in cubosomal nanoparticles and studied its anticancer effects in vitro and in vivo. Methods: The TQ-loaded cubosomes were prepared by the emulsification technique. To study the cytotoxic and anticancer effects of the formulations, we performed MTT and trypan blue exclusion assays on human breast cell lines. These included the nontumorigenic MCF-10A mammary epithelial cells, the aggressive MDA-MB-231 and the MCF-7 breast cancer cell lines. These experiments were complemented with apoptosis assays in MDA-MB-231 cells by detecting caspase 3 cleavage and morphological changes in nuclei of treated cells by DAPI staining. Finally, the cell-NP interaction and the uptake of the formulation into the cells were investigated to determine their mode of entry and subcellular localization. To determine whether TQ-loaded cubosomes confer an advantage over free TQ, 6 to 8-week-old female immunocompromised NSG mice (n=6 per group) were xenografted with MDA-MB-231 breast cancer cells. Once tumors were established, treatment started with blank cubosomes, or with 15 mg-kg of free TQ or its equivalent in TQ-loaded cubosomes. The body weight of mice and volume of tumors were measured three times-week and two animal experiments were performed. In the first experiment, treatment was administered intraperitoneally for 21 days. In the second experiment, treatment was administered subcutaneously to avoid the side effects of ascites formation that was observed with intraperitoneal injections. Results: We observed a dose-dependent inhibitory response to treatment with TQ or TQ-loaded cubosomes in both breast cancer cell lines. According to MTT assay, at concentrations up to 30 μM, TQ-loaded cubosomes h