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Uptake, delivery, and anticancer activity of thymoquinone nanoparticles in breast cancer cells

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Abstract

Thymoquinone (TQ) is a promising anticancer molecule but its development is hindered by its limited bioavailability. Drug encapsulation is commonly used to overcome low drug solubility, limited bioavailability, and nonspecific targeting. In this project, TQ nanoparticles (TQ-NP) were synthesized and characterized. The cytotoxicity of the NP was investigated in nontumorigenic MCF-10-A breast cells, while the uptake, distribution, as well as the anticancer potential were investigated in MCF-7 and MDA-MB-231 breast cancer cells. Flash Nanoprecipitation and dynamic light scattering coupled with scanning electron microscopy were used to prepare and characterize TQ-NP prior to measuring their anticancer potential by MTT assay. The uptake and subcellular intake of TQ-NP were evaluated by fluorometry and confocal microscopy. TQ-NP were stable with a hydrodynamic average diameter size around 100 nm. Entrapment efficiency and loading content of TQ-NP were high (around 80 and 50 %, respectively). In vitro, TQ-NP had equal or enhanced anticancer activity effects compared to TQ in MCF-7 and aggressive MDA-MB-231 breast cancer cells, respectively, with no significant cytotoxicity of the blank NP. In addition, TQ and TQ-NP were relatively nontoxic to MCF-10-A normal breast cells. TQ-NP uptake mechanism was both time and concentration dependent. Treatment with inhibitors of endocytosis suggested the involvement of caveolin in TQ-NP uptake. This was further confirmed by subcellular localization findings showing the colocalization of TQ-NP with caveolin and transferrin as well as with the early and late markers of endocytosis. Altogether, the results describe an approach for the enhancement of TQ anticancer activity and uncover the mechanisms behind cell-TQ-NP interaction.

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Acknowledgments

This work was funded by the Swedish Research Council (Swedish Research Links 2013-6651) and the FAS Dean’s office of the American University of Beirut, Beirut, Lebanon. We are very grateful to the members of the Central Research Science Laboratory at the American University of Beirut for their technical assistance. Finally, we would like to acknowledge Ms Dana Fakhreddine and Mr Elia Salibi for their help in some experiments of this project. This work was funded by the Swedish Research Council (Swedish Research Links 2013-6651) and the Faculty of Arts and Sciences Dean’s office of the American University of Beirut, Beirut, Lebanon.

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Authors and Affiliations

  1. Department of Biology, American University of Beirut, Beirut, Lebanon

    Isabelle Fakhoury & Hala Gali-Muhtasib

  2. Department of Chemical and Petroleum Engineering, American University of Beirut, Beirut, Lebanon

    Walid Saad

  3. Department of Chemistry, American University of Beirut, Beirut, Lebanon

    Kamal Bouhadir

  4. Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology, Uppsala University, Uppsala, Sweden

    Peter Nygren

  5. Experimental Tumor Pathology, Institute for Pathology, University of Erlangen-Nuremberg, Erlangen, Germany

    Regine Schneider-Stock

  6. Department of Anatomy, Cell Biology, Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon

    Hala Gali-Muhtasib

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  1. Isabelle Fakhoury
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Corresponding author

Correspondence to Hala Gali-Muhtasib.

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11051_2016_3517_MOESM1_ESM.tif

Supplementary Fig. 1. A. Bar graph showing the concentration-dependent uptake of NR-loaded TQ-NP expressed as the average of the mean fluorescence intensity of MCF-7 and MDA-MB-231 cells ± SE. The data represent two independent experiments (n = 3). * indicates p < 0.05 with respect to the control, † indicates p < 0.05 with respect to cells treated with 10 μg/ml B. Confocal microscopic images of MCF-7 and MDA-MB-231 cells after 30 min incubation at 37 °C with 25 μg/ml and 50 μg/ml NR-loaded TQ-NP respectively. The nuclei are stained with Hoechst (0.5 μg/ml) (blue). The uptake of NP (green) was visualized by overlaying images obtained by NR filter and Hoechst filter using a Zeiss 710 confocal microscope and a ×63 oil objective. Bar = 5 μm. C. Bar graph showing the uptake of NR-loaded TQ-NP over time expressed as the average of the mean fluorescence intensity of MCF-7 and MDA-MB-231 cells ± SE. The data represent two independent experiments (n = 3). * indicates p < 0.05 with respect to the control, † indicates p < 0.05 with respect to 5 min incubation time and ¥ indicates p < 0.05 with respect to 15 min incubation time. (TIF 1312 kb)

11051_2016_3517_MOESM2_ESM.tif

Supplementary Fig. 2. The intracellular distribution of NR-loaded TQ-NP. MCF-7 breast cancer cells were incubated with 25 μg/ml of NR-loaded TQ-NP in growth medium for 30 min before preparation for incubation with the primary antibodies of the different endocytic markers (dilution 1:100). Caveolin, transferrin, EEA-1 and Lamp-1 are visualized in red, the NP in green and the nuclei stained with Hoechst, are shown in blue. The images were obtained using a Zeiss 710 confocal microscope and a ×63 oil objective. Bar = 5 μm.  (TIF 1312 kb)

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Fakhoury, I., Saad, W., Bouhadir, K. et al. Uptake, delivery, and anticancer activity of thymoquinone nanoparticles in breast cancer cells. J Nanopart Res 18, 210 (2016). https://doi.org/10.1007/s11051-016-3517-8

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