The Effects Of Multi-wall Carbon Nanotube And Its Contained Fe⁺⁺ Effects On PC12Cells

Objective: To investigate the effects of Multi-wall carbon nanotube and its contained Fe⁺⁺on morphology of PC12cells.Methods:ICP-MS was used to precisely detect the content of Fe⁺⁺in multi-wall carbon nanotubes;There are three test groups including control group，high-Fe⁺⁺MWCNTs group and Low-Fe⁺⁺MWCNTs group. CCK-8assay was used to detect the effect of different concentrations ofMWCNTs （5,10,30,60μg/ml） on cell viability of PC12cells at different times（24,48,72h）.Dead/live staining was used to detect the state of PC12cells48h after incubated withmulti-wall carbon nanotubes. Immunofluorescence assay was used to detect theconformational change of F-actin. To observe the effects of MWCNTs on differentiation ofPC12cells, PC12cells were incubated with MWCNTs for48h and continuously induced by50ng/ml NGF for7days, then the number of neuritis per cell and differentiation ratio ofPC12were counted; Immunofluorescent assay was to detect conformation and distribution ofcell specific sytoskeletal protein β-tubulin, and the expression of TH. To explore themechanism of effect of multi-wall carbon nanotubes on morphology of PC12cells48h afterincubated with MWCNTs, TEM was used to localize the multi-wall carbon nanotubes in celland changes of cell ultrastructure; confocal laser scanning microscopy was used detect theproduction of intracellular ROS.Results:ICP-MS assay showed the content of iron High-Fe⁺⁺ MWCNTs was12%and Low-Fe⁺⁺MWCNTs was4.2%; CCK-8assay showed the cell viability did not change with differentconcentrations of mutil-wall carbon nanotubes（5,10,30,60μg/ml） incubating for24h or48h,while cytotoxicity was detected72h after incubated with more than10μg/ml High-Fe⁺⁺ MWCNTs or more than30μg/ml Low-Fe⁺⁺MWCNTs;Immunofluorescence assay showedthat cells2days after incubated with30μg/ml High-Fe⁺⁺MWCNTs decreased express levelof F-actin in cell edges or condenses into nodules kind, Low-Fe⁺⁺MWCNTs abated thetension of F-actin.When cells were incubated with multi-wall carbon nanotubes for48h following with50ng/ml NGF continuously induction cell differentiation for7days,we observed that High-Fe⁺⁺MWCNTs and Low-Fe⁺⁺MWCNTs have no effects on number of neuritis per cells anddifferentiation rate, however，both inhibited the expression of TH; High-Fe⁺⁺MWCNTsinhibited expression of β-tubulin Ⅲ, and weaken the overlap of F-actin andβ-tubulinⅢinapophysis.The preliminary study on the effect of carbon nanotubes on cells showed both high-Fe⁺⁺MWCNTs and low-Fe⁺⁺MWCNTs could enter into cells and located in cell vacuoles withfold-like or aggregated state. PC12cells of high-Fe⁺⁺MWCNTs group trended obviouslyapoptosis, the production of ROS in cells was small.Conclusions:1. Incubation with high-Fe⁺⁺MWCNTs for72h caused a dose-dependent inhibitedproliferation in PC12cells investigated by CCK-8assay. We also analyzed the viability oftreatment of PC12cells with low-Fe⁺⁺MWCNTs.From the results of the data,weobtained that over than30ug/ml low-Fe⁺⁺MWCNTs resulted in an increased cytotoxicity.2. High-Fe⁺⁺MWCNT inhibit conformational changes of F-actin and decrease theexpression of skeleton proteinβ-tububinⅢand distribution trend to axons.3. Both high-Fe⁺⁺MWCNTs and Low-Fe⁺⁺MWCNTs inhibit the expression of TH.4. High-Fe⁺⁺MWCNTs localize in cells with fold-like or aggregated state, lead to the cellstrend to apoptosis.