Thymoquinone poly (lactide-co-glycolide) nanoparticles exhibit enhanced anti-proliferative, anti-inflammatory, and chemosensitization potential

Abstract

Thymoquinone (TQ), derived from the medicinal spice Nigella sativa (also called black cumin), has been shown to exhibit anti-inflammatory and anti-cancer activities. In this report we employed polymer-based nanoparticle approach to improve upon its effectiveness and bioavailability. TQ was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and the stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy confirmed particle diameter between 150 and 200nm. Electrophoretic gel shift mobility assay showed that TQ nanoparticles (NP) were more active than TQ in inhibiting NF-kappaB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), those are markers of cell proliferation, metastasis and angiogenesis, respectively. TQ-NP were also more potent than TQ in suppressing proliferation of colon cancer, breast cancer, prostate cancer, and multiple myeloma cells. Esterase staining for plasma membrane integrity revealed that TQ-NP were more potent than TQ in sensitizing leukemic cells to TNF- and paclitaxel-induced apoptosis. Overall our results demonstrate that encapsulation of TQ into nanoparticles enhances its anti-proliferative, anti-inflammatory, and chemosensitizing effects.

Figure 1

Design and characterization of thymoquinone nanoparticles. (A) Structure of thymoquinone. (B) Thymoquinone nanoparticles: Morphology by scanning electron microscopy (SEM) and details of the polymer used for the preparation of thymoquinone nanoparticles. (C) Drug release curve for thymoquinone nanoparticles. (D) Nanoparticle size was determined by transmission electron microscopy (TEM) after mechanical extrusion using membrane extruder. The particle size were ranged between 150–200nm. (E) Distribution of the nanoparticles was measured by using microsampler attached photon correlation spectroscopy. R, radius.

Figure 2

Effect of thymoquinone and thymoquinone nanoparticles on cell proliferation and apoptosis in myeloid leukemia (KBM-5) cells. (A) Five thousand cells, per well were seeded in triplicate onto 96-well plates; treated with different concentrations of TQ and TQ-NP for 72 h; measured cell viability by the MTT method and presented as percent cell viability. Data are the representative of three independent experiments. Points, mean (n = 3); bars, S.E. *, P < 0.05; **, P < 0.01 versus untreated cells. (B) Cells were treated with 10 μM TQ or TQ-NP for 8 h and the cells were then treated with TNF (1 nM) for 24 h. Cells were stained with PI/annexin V and analyzed by flow cytometry.

Figure 3

Effect of thymoquinone and thymoquinone nanoparticles on cell proliferation in different tumor cells. Five thousand human colon cancer (HCT-116), breast (MCF-7), prostrate (PC-3) and multiple myeloma (U-266) cells, per well were seeded in triplicate onto 96-well plates; treated with the each compound at 10 μM for 72 h; measured cell viability by the MTT method and presented as percent cell viability. Data are the representative of three independent experiments. Columns, mean (n = 3); bars, S.E. Data are the representative of three independent experiments. Points, mean (n = 3); bars, S.E. *, P < 0.05; **, P < 0.01, ***, P < 0.001 versus untreated cells.

Figure 4

Effect of thymoquinone nanoparticles on TNF-induced NF-κB activation. (A) KBM-5 (1 × 10⁶ cells/mL) cells were treated with indicated concentrations of thymoquinone or thymoquinone nanoparticles for 4 h then co-incubated with TNF (0.1 nM) for 30 min. Nuclear extracts were prepared and the NF-κB activity was examined by EMSA (panel A). (B) Relative NF-κB activation by TNF and its inhibition by TQ and TQ-NP. Data are the representative of three independent experiments. Points, mean (n = 3); bars, S.E. *, P < 0.05; **, P < 0.01 versus untreated cells.

Figure 5

Effect of thymoquinone nanoparticles on the expression of TNF-induced NF-κB-regulated gene products. KBM-5 (1 × 10⁶ cells/mL) cells were co-incubated with indicated concentration of TQ or TQ-NP and TNF (1 nM) for 16 h. The cells were harvested and analyzed for the expression of cyclin D1, MMP-9, and VEGF by western blot. β-actin was used as a loading control. Densitometric values of bands were corrected based on β-actin and expressed relative to that of untreated cells, which was set at 1.0.

Figure 6

Effect of thymoquinone nanoparticles on sensitization of tumor cells to TNF and paclitaxel. KBM-5 (1 × 10⁶ cells/mL) cells were incubated with 10 μM of TQ and TQ-NP for 4h. The cells were treated with TNF (1 nM) or paclitaxel (5 nM) for 16 h and were harvested and stained with Live/Dead assay reagent as per the manufacturer’s protocol as described in methods.