Functionalized Multi-walled Carbon Nanotubes And L02 Cell Interaction

Nano-materials have abundant potential applications on many fields due to their excellent physical and chemical properties. However, some preliminary studies showed that many nano-scale materials had potential toxic effects, so it is very necessary to evaluate the biological effects and toxicity mechanisms of nano-materials on the cellular and molecular level. Functionalized multi-walled carbon nanotubes (MWCNT) may have a huge application in biomedicine field because of their unique properties. The bio-safety of MWCNTs also has been drawing increasing attention. However, the toxicity and biocompatibility of MWCNTs have not been thoroughly investigated, many results are even contradictory. The functionalized MWCNTs can enter into the organism circulatory system when exposed to MWCNTs through inhalation, orally take or intravenous injection. Because liver have first-pass effects and some data show that nano-materials mainly accumulate in the liver, the observation of the effects of carbon nanotubes on liver cells is very important. In this study, we chose the human normal L02cell line as the model cell and investigated the interactions between MWCNTs and L02cells, thus to give a theoretical basis and experiment foundation for the further study of the biological effects of nano-materials.The study includes the following parts:(1) Multi-walled carbon nanotubes (MWCNT), carboxyl multi-walled carbon nanotubes (MWCNT-COOH) and hydroxyl multi-walled carbon nanotubes (MWCNT-OH) were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). TEM showed that the average outside diameters (10to20nm) and the average length (10to30μm) of the three MWCNTs were similar. SEM indicated that the three MWCNTs had a similar surface topography. XPS demonstrated that the MWCNT-COOH and MWCNT-OH had relatively high peak area at289ev and286ev, respectively, indicating that they have been modified by carboxyl and hydroxyl groups respectively. The metal catalysts impurities were not detected and the sample purity is greater than98%. (2) To investigate the effects of MWCNTs on the cytotocixity of L02cells. The carbon nanotubes at concentrations of12.5,25,50,100and200μg/ml were incubated with L02cells for24,48and72h, respectively. The cell viability was evaluated by WST assay and Celltiter-Glo luminescent cell viability assay. The two assays showed that the cells toxicity was caused in a time-and dose-dependent manner. MWCNT-COOH was less cytotoxic as compared to MWCNT which demonstrated to be similarly cytotoxic with MWCNT-OH. Modification of MWCNT with carboxyl group and hydroxyl group improved the biocompatibility of MWCNTs to some extents. Analysis from the cell mophology and nuclear Hoechst33342staining observation and apoptosis/necrosis revealed that MWCNTs could induce apoptosis in L02cells in a time-and dose-dependent manner. Primary MWCNTs compared with functionalized MWCNTs are more likely to induce cell apoptosis。(3) To investigate the effect of MWCNTs on the oxidative stress, membrane integrity and cell cycle of L02cells. MWCNTs induced the intercellular reactive oxygen species (ROS) formation and intracellular antioxidant GSH eliminated the intracelluar ROS significantly at24h after treated with200μg/mL MWCNTs. But the GSH decreased slowly after48h shows that the L02cells had be induced oxidative stress. The increase leakage of LDH demonstrated that oxidative stress destroyed the membrane integrity of L02cells in a time-and dose-dependent manner. MWCNTs induced G0/G1phase arrested slightly in L02Cells after treatment with MWCNTs for24h. Compared to the control group, the ratio in G0/G1phase increased slightly, and the ratio in G2/M phase decreased. The result shows that the MWCNTs can arrest cell cycle at G0/G1phase. This might be one of the mechanisms contributing to the cytotoxicity of MWCNTs even though it is not the only mechanism involved.(4) To investigate the effect of MWCNTs on the mitochondria and apoptosis signal pathway of L02cells. MWCNTs induced L02cells apoptosis was associated with the decrease of mitoehondrial membrane potential (MMP) and the secretion of cyto-c. MWNTs could activate caspases-9,-3. MWCNTs could also activate the death receptor apoptosis pathway caspases-8. These results suggest that L02cells apoptosis induced by MWCNTs may be through mitochondria dependent pathway and also death receptor dependent pathway. We also found that the functionalized MWCNTs have the less ability to induce cell apoptosis from mitochondria dependent pathway. This might be one of the reasons that the f-MWCNTs have better biocompatibility than primary MWCNTs.