Study On Cytotoxicity Of Functionalized Multi-walled Carbon Nanotubes (f-MWNTs)

OBJECTIVES:In the past years,carbon nanotubes(CNTs)have been intensively investigated for its wide potential applications especially in biomedicine because of the unique physicochemical properties. However,carbon nanotubes have raised several questions about their safety and environmental impact.Research on the toxicity of carbon nanotubes has just begun and the data are still fragmentary and subject to criticisms.Surface modification of CNTs has considerable impact on their reactivity.A functionalized multi-walled carbon nanotubes(f-MWNTs)library has been synthesized for the first time in our group.In order to find out the dose-response relationship relevant with the physicochemical properties of MWNTs,study on the cytotoxicity of the f-MWNTs in the library was performed.Four kinds of f-MWNTs with different cytotoxicity(MWNTs-002, MWNTs-003,MWNTs-004 and MWNTs-005)have been selected for further research after the primary cytotoxicity screening.METHODS:Two cell lines,NHFB and HepG2,have been chosen for the primary cytotoxicity screening of f-MWNTs library.The concentration of f-MWNTs is 200μg/mL.In the experiment,we used microscopy to observe the cell morphology change and WST-1 assay to evaluate cell survival.MWNTs-002,MWNTs-003,MWNTs-004 and MWNTs-005 were chosen for the following studies.Firstly,cell uptake and intracellular localization of f-MWNTs was imaged by transmission electron microscopy(TEM).Secondly,Cellular reactive oxygen species(ROS)was determined by fluorometry with DCF as the fluorescence probe.Finally,cell cycle assay was performed with flow cytometry.RESULTS: 1.Cytotoxicity research of f-MWNTs libraryThe effects of f-MWNTs appeared dose dependent,but not time dependent.The dispersibility of f-MWNTs with different surface modification is very different,and their cytotoxicity is also different. Cell viability after treatment with f-MWNTs is about above 50%. MWNTs-002 has the greatest effect on cell viability with 53%for NHFB,and 60%for HepG2,respectively.2.Cell morphology observationCell morphology changed a lot after f-MWNTs treatment.In normal conditions,NHFB is fibriform,but the cells exposed to f-MWNTs are very thin,cell morphology changed to filiform.Cell adhesion and spread ability are very poor.With smaller cell number compared to control group,f-MWNTs inhibit cell proliferation obviously.3.Intracellular localization of f-MWNTsMWNTs-002,MWNTs-0O3,MWNTs-004 and MWNTs-005 locate within the intracytoplasmic vacuoles,and do some damage to the cells.For example,the structure of cell membrane was destroyed, euchromatin reduced and apoptotic bodies were produced etc.4.Determination of intracellular reactive oxygen species(ROS)F-MWNTs can enhance the intracellular ROS in HepG2 cells.This response shows a dose-dependent manner.When the concentration of f-MWNTs is above 50μg/mL,cellular ROS increased noticeably. However,the level of cellular ROS decreased along with the treatment time.5.Cell Cycle(CC)F-MWNTs can induce G0/G1 phase arrest slightly in HepG2 cells after treatment with MWNTs-002 for 12 h.Compared to the control group,the ratio in G0/G1 phase increased slightly,and the ratio in G2/M phase decreased.At 200μg/mL,the ratio in G0/G1 phase is 65.86%,while in control group,it is 57.45%.When the cells exposed to f-MWNTs for 12 h,G0/G1 phase arrest got to a vertex.CONCLUSIONS: 1.The effects of f-MWNTs are dose dependent,but not time dependent.The cytotoxic response of cells in culture is dependent on the surface modification of MWNTs.2.F-MWNTs can inhibit cell proliferation,but not induce cell death.3.F-MWNTs can cross cell membrane,locate in vacuoles in cytoplasm and damage to the cells.4.F-MWNTs can increase the cellular ROS.5.F-MWNTs can induce G/G1 phase arrest slightly.