Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines

Abstract

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.

Fig 1. Transmission electron microscopy images of nanoparticles.

Images of (A) diamond (ND) and (B) graphite (NG) nanoparticles. Scale bar: 100 nm.

Fig 2. Glioblastoma and hepatoma cells morphology.

U87, U87-EGFP, C3A and C3A-EGFP cells morphology after 2 h exposure to medium containing nanoparticles at the highest concentration: graphite 100 μg/mL (NG 100) and diamond 100 μg/mL (ND 100), nontreated cells were used as a control (Control). Analysis of the cells morphology was performed using an inverted fluorescence microscope. Images of the labeled cells, U87-EGFP and C3A-EGFP, were captured using a green fluorescence filter. Scale bar: 200 μm.

Fig 3. MTT test analysis.

U87, U87-EGFP, C3A and C3A-EGFP cells metabolic analysis was performed after 2 and 24 h of exposure to the culture medium containing nanoparticles at different concentrations: diamond 20 μg/mL (ND 20), 50 μg/mL (ND 50) and 100 μg/mL (ND 100), and graphite 20 μg/mL (NG 20), 50 μg/mL (NG 50) and 100 μg/mL (NG 100); nontreated cells were used as a control (Control). Metabolic activity of cells, expressed as I _CV, after 2 h of exposure to the culture medium with the above-mentioned different concentrations of diamond and graphite nanoparticles: U87 and U87-EGFP (A), and C3A and C3A-EGFP (B); and after 24 h of exposure to the culture medium with above-mentioned different concentrations of diamond and graphite nanoparticles: U87 and U87-EGFP (C), and C3A and C3A-EGFP (D). Data analysis was carried out by two-way ANOVA and the differences between the groups were tested by Duncan’s test. Data were averaged from three replicates (n = 3); P < 0.05. No significant interaction (nanoparticles with transduction) was observed.

Fig 4. Glioblastoma and hepatoma cells status (CI).

U87, U87-EGFP, C3A and C3A-EGFP CI monitored by a real-time cell analyzer (RTCA) after 1 and 2 h of exposure to the culture medium containing graphite and diamond nanoparticles at different concentrations: diamond 20 μg/mL (ND 20), 50 μg/mL (ND 50) and 100 μg/mL (ND 100), and graphite 20 μg/mL (NG 20), 50 μg/mL (NG 50) and 100 μg/mL (NG 100); nontreated cells were used as a control (NT). (A) U87 (blue lines) and C3A cells (red lines) treated with graphite (NG) after 1 h (vertical blue marker line) and 2 h (vertical red marker line) and control (vertical green marker line); (B) U87-EGFP cells (violet lines) and C3A-EGFP cells (green lines) treated with graphite after 1 h (vertical blue marker line) and 2 h (vertical red marker line) and control (vertical green marker line); (C) U87 (blue lines) and C3A cells (red lines) treated with diamond (ND) after 1 h (vertical blue marker line) and 2 h (vertical red marker line) and control (vertical green marker line); (D) U87-EGFP cells (violet lines) and C3A-EGFP cells (green lines) treated with diamond (ND) after 1 h (vertical blue marker line) and 2 h (vertical red marker line) and control (vertical green marker line). Data analysis was carried out by two-way ANOVA and the differences between the groups were tested by Duncan’s test. Data were averaged from three replicates (n = 3); P< 0.05. No significant interaction (nanoparticles with transduction) was observed.

Fig 5. BrdU incorporation test.

U87, U87-EGFP, C3A and C3A-EGFP cells proliferation (expressed as I _CP) analysis was performed 24 h after treatment with the culture medium containing nanoparticles at different concentrations: diamond 20 μg/mL (ND 20), 50 μg/mL (ND 50) and 100 μg/mL (ND 100), and graphite 20 μg/mL (NG 20), 50 μg/mL (NG 50) and 100 μg/mL (NG 100); nontreated cells were used as a control (Control). (A) U87 and U87-EGFP cells and (B) C3A and C3A-EGFP cells after 24 h of exposure to the medium with different concentrations of diamond and graphite nanoparticles. Data analysis was carried out by two-way ANOVA and the differences between the groups were tested by Duncan’s test. Data were averaged from three replicates (n = 3); P< 0.05. We observed significant interactions (nanoparticles with transduction) in glioblastoma cells (P = 0.0001).

Fig 6. Human serum albumin concentration in culture medium.

Albumin secreted by the C3A and C3A-EGFP cells treated for 24 h with the culture medium containing nanoparticles at different concentrations measured by ELISA test: diamond 20 μg/mL (ND 20), 50 μg/mL (ND 50) and 100 μg/mL (ND 100), and graphite 20 μg/mL (NG 20), 50 μg/mL (NG 50) and 100 μg/mL (NG 100). Albumin secretion by cells after 24 h exposure to the medium with different concentrations of diamond and graphite nanoparticles: (A) C3A cells and (B) C3A-EGFP cells. Nontreated cells were used as a control (Control). Data were analyzed using a two-way ANOVA with the Duncan’s test. Data were averaged from three replicates (n = 3); P < 0.05. No significant interaction (nanoparticles with transduction) was observed.