Preparation Of Water-soluble Carbon Nanotubes And Preliminary Study Of Their Toxicity To Human Embryonic Kidney Cells (HEK-293T) And Liver Cancer Cells (HepG₂)

Objective:To attach Carboxyl groups to the sidewall of carbon nanotubes treated by mixture of concentrated nitric acid and concentrated sulfuric acid to realize functionalization and modi—fication of carbon nanotubes. The contents of Carboxyl groups in the surface of carbon nanotubes was analyze by thermo gravimetric analysis(TGA). Then attach PEG to the surface of MWCNTS-COOH. To investigate the cytotoxic effect of functionalized multi-walled carbon nanotubes (MWCNTs-PEG) on human embryo kidnet cell(293T) and human hepatoma cell line(HepG2).Method:1. Carboxyl groups functionalized MWNT (MWNT-COOH) was prepared by mixture of concentrated nitric acid and concentrated sulfuric acid. The contents of Carboxyl groups in the surface of carbon nanotubes was analyze by thermo gravimetric analysis(TGA).2. MWNT-COOH was reaction with dimethylformamide(DMF) to get acyl chlorination products. This acyl chlorination products then reaction with polyethyleneglycol(PEG) to get functionalized MWNTs contains the hydrophilic groups(-PEG). Finally get product MWCNTs-PEG.3. MWCNTs-PEG was soluble in Cell Medium (DMEM). The carbon nanotubes at concentrations of6.25,12.5,25,50,100and200ug/ml were incubated with human embryo kidnet cell (293T) and human hepatoma cell line(HepG2) for24,48and72hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay(MTT).4. MWCNTs-PEG solution at concentrations of6.25,12.5,25,50,100and200ug/ml were incubated with human hepatoma cell for24h.Death cells was dyeing with propidium iodide (PI).The cell mortality was determined by flow cytometry(FCM).Result:1.The carboxyl groups was attach to the original multi-walled carbon nanotubes by mixture of concentrated nitric acid and concentrated sulfuric acid. The contents of Carboxyl groups in the surface of carbon nanotubes was analyze by thermo gravimetric analysis. To estimate the Carboxyl groups contents is2.64%2. When the hydrophilic groups(PEG) was attached to the MWNT-COOH, the solubility of MWNTs was increased. Put it dissolve in phosphate buffer(PBS) and treated by ultrasound can form stable black solution.3. The solution of multi-walled carbon nanotubes contains hydrophilic groups of polyethyleneglycol were incubated with human embryo kidnet cell (293T) and human hepatoma cell line(HepG2) for24hours. Along with the increase of concentration, the cell survival rate down gradually, indicate the cytotoxic exist concentration dependence relationship. The viabilities of cells comparison with control, difference was statistically significant(P<0.05) in50ug/ml?100ug/ml and200ug/ml this three groups. The groups of concentration?100ug/ml toxicity grade is I,Groups of200ug/ml the toxicity grade is II. In48and72hours the toxicity no further change.4. Dyeing with propidium iodide (PI) and watch under Laser Scanning Confocal Microscope can see Death cells show strong red fluorescence, but normal cells not dyeing. Along with the increase of concentration, the death cells gradually increased. The viabilities of cells in100ug/ml and200ug/ml this two groups comparison with control, difference was statistically significant(P<0.05) detected by flow cytometry(FCM).Conclusion1. The solubility of MWNTs was increased by attach hydrophilic groups(PEG) to the surface of multi-walled carbon nanotubes.2. The cytotoxic of MWNTs-PEG exist concentration dependence relationship. Along with the increase of concentration, the cell survival rate down gradually.3.Based on ISO2109932-5cytotoxic standard, MWNTs-PEG concentration?100ug/ml toxicity grade is?,consider it's no toxicity. When concentration is200ug/ml, toxicity grade is ?, consider has slight toxicity. In the experiment observation time found no change in toxicity grade.