Preparation and characterization of realgar nanoparticles and their inhibitory effect on rat glioma cells

Abstract

Aim: Our objective was to prepare a new nano-sized realgar particle and characterize its anti-tumor effect on tumor cells.

Methods: Nanoparticles were prepared by coprecipitation and were detected by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry (EDS), and dynamic light scattering. An anti-proliferative effect of realgar nanoparticles on rat glioma (C6) cells was determined by the MTT assay. Cell cycle and apoptosis rates were observed by flow cytometry. Apoptosis-related gene expression was detected by immunofluorescence staining.

Results: Realgar nanoparticles were successfully prepared. The particles were spherical, with an average diameter of approximately 80 nm, and contained arsenic and sulfur elements. Realgar nanoparticles inhibited C6 cell proliferation and induced apoptosis in a dose- and time-dependent manner. Treatment of C6 cells with realgar nanoparticles significantly increased the proportions of cells in S and G2/M phases, decreased the proportion of cells in G0/G1 phase, downregulated Bcl-2 expression, and substantially upregulated Bax expression.

Conclusion: Realgar nanoparticles significantly inhibited C6 glioma cell proliferation and promoted cell apoptosis by inducing the upregulation of Bax and downregulation of Bcl-2 expression. Realgar nanoparticles are a promising in vitro anti-cancer strategy and may be applicable for human cancer therapy studies.

Keywords: characterization; inhibitory effect; preparation; realgar.

Figure 1

Transmission electron micrograph of realgar nanoparticles.

Figure 2

Hydrodynamic diameter distribution of realgar nanoparticles.

Figure 3

Energy dispersive spectrometric determination of realgar nanoparticle elemental composition.

Figure 4

Cytotoxicity of realgar nanoparticles, purified realgar and traditional realgar at 24 hours (A), 48 hours (B), 72 hours (C) in C6 cells. Note: Each data point shown is the mean ± SD from three independent experiments.

Figure 5

Realgar treatment altered the cell cycle distribution. C6 cells treated with realgar nanoparticles, purified realgar, traditional realgar, or PBS for 24 h were stained with PI and analyzed by flow cytometry (A). Graphical representation of realgar-treated cells in different phases of cellular cycle. Realgar treatment decreased the percentage of cells in G0/G1 and increased the percentages of cells in G2/M and S phases (B). Notes: Results represent means ± SD of three independent experiments.

Figure 6

Realgar treatment promoted apoptosis. C6 cells were treated with realgar nanoparticles, purified realgar, traditional realgar, or PBS for 24 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry (A). Realgar treatment increased the percentage of apoptotic cells (B). Note: Results represent means ± SD of three independent experiments.

Figure 7

Immunofluorescence staining of Bcl-2 and Bax proteins in C6 cells treated with PBS (A), traditional realgar (B), purified realgar (C), or realgar nanoparticles (D). Semi-quantitative determination of the percentage of Bcl-2-positive or Bax-positive cells (E). Notes: Magnification, ×400. Experiments were repeated in triplicate. For each sample, the percentage of positive cells was determined by counting four representative areas under the fluorescence microscope.