The Cytotoxicity And Underlying Mechanism Of Gold Nanorods In A549 Cells

Gold nanorods(AuNRs)are a kind of rod-like gold nanoparticles which with the scale between 1 and 100 nm.Because of their unique optical properties,quantum tunneling effects,biocompatibility,surface ease of modification and stability,AuNRs have been considered to be as one of the most promising nanomaterials.In recent years,with the development of nanotechnology applications in the biomedical field,there are more and more reports about applications of AuNRs in cell and animal imaging,drug and gene delivery,diagnosis and treatment of a variety of diseases.However,with the widespread use of AuNRs,people are increasingly exposed to AuNRs,and the biosafety problems caused by exposure to AuNRs are also receiving increasing attention.Therefore,it is should to explore the potential impact of AuNRs on organisms and their underlying mechanism for more safer and widely used.In this study,firstly,A549 cells were treated with different concentrations of AuNRs for 6h,12 h and 24 h,and then the effects of AuNRs on the viability and cell membrane damage of A549 cells were observed by CCK-8 and LDH test.On the basis of this,the effects of AuNRs on the ultrastructure of the cells were observed by transmission electron microscopy,and the cytotoxicity of AuNRs was evaluated.Furthermore,western blot technique,laser scanning confocal microscopy and other molecular biology methods were used to observe whether the cytotoxicity of AuNRs was related to the damage mitochondrial-related functions that promote oxidative stress and induce cell autophagy.The related experimental results were as follows:I.The effects of AuNRs on the toxicity of A549 cellsFirst,cultured A549 cells were treated with different concentrations of AuNRs(0μg/ ml,0.5μg/ml,1μg/ml,2μg/ml,4μg/ml)for 6h,12 h and 24 h respectively,cell viability,cell membrane damage and cell ultrastructure were then observed,the experimental results are as follows:1.The effects of AuNRs on the viability of A549 cellsThe results of CCK-8 showed that the viability of A549 cells was decreased in a dose-dependent and time-dependent manner after treatment with different concentrations of AuNRs.It was suggested that the cytotoxicity of AuNRs was dose-dependent and time-dependent.2.The effects of AuNRs on the membrane damage of A549 cellsThe results of LDH test showed that LDH leakage and supernatant LDH increased in a dose-dependent and time-dependent manner after treatment with different concentrations of AuNRs.It was suggested that the cytotoxicity of AuNRs was dose-dependent and time-dependent.The higher the concentration of AuNRs,the longer the time of AuNRs exposure,the greater the toxicity of AuNRs.3.The effects of AuNRs on the ultrastructure of A549 cellsThe results of transmission electron microscopy showed that AuNRs were able to enter A549 cells and existed in the cytoplasm and partial membrane vesicles in the form of individual particles and aggregates,but no AuNRs were observed in the nucleus.In addition,some of the mitochondria in the cells were swollen to varying degrees,and the number of autophagosomes was significantly increased than that of the control group.II.The effects of AuNRs on autophagy of A549 cellsIt has been found that autophagy is one of the important mechanisms of cytotoxicity of nanomaterials.So we wondered to know whether the toxicity of AuNRs to A549 cells is related to autophagy? Therefore,we conducted the following study,the experimental results are as follows:1.The effects of AuNRs on LC3 in A549 cellsThe results of laser scanning confocal microscopy showed that the expression of LC3 in A549 cells treated with different concentrations of AuNRs increased in a dose-dependent manner.Among them,LC3 expression of Con group mainly concentrated in the nucleus.With the increase of the concentration of AuNRs,the expression of LC3 was gradually transferred from the nucleus to the cytoplasm,and the expression of LC3 in the nucleus gradually decreased and vacuolated.Furthermore,A549 cells were treated with 2μg/ml of AuNRs for 0h,6h,12 h and 24 h,respectively.The results showed that the expression of LC3 in A549 cells increased in a time-dependent manner.LC3 expression of Con group mainly concentrated in the nucleus.With the increase of the exposure time of AuNRs,the expression of LC3 was gradually transferred from the nucleus to the cytoplasm,and the expression of LC3 in the nucleus gradually decreased and vacuolated.2.The effects of AuNRs on autophagy-related proteins in A549 cellsWestern blot results showed that the expression of LC3-II,ATG4,ATG16 and Beclin1 protein was significantly increased after treatment of A549 cells with different concentrations of AuNRs for 6h,while the expression of P62,a kind of autophagic substrate protein,was significantly decreased.It is suggested that AuNRs induce increased of autophagy in A549 cells in the dose and time-dependent manner.3.CQ can inhibit the autophagy induced by AuNRsWestern blot results showed that under normal culture conditions,the expression of ATG16 and LC3-II in Con group was in a low level.The expression of ATG16 and LC3-II was significantly higher than that of Con group after CQ treatment,indicating that CQ could inhibit the degradation of basal autophagosomes level.The expression of ATG16 and LC-II was significantly increased compared with the control group after administration of AuNRs.AuNRs and CQ co-treated A549 cells for 6h,high expression of ATG16 can be reversed by CQ,but LC3-II expression is further increased.This result indicates that CQ can not only inhibit the initiation of autophagy by AuNRs,but also inhibit autophagosomal degradation.III.Oxidative stress mediated autophagy induced by AuNRs in A549 cellsIn recent years,studies have found that oxidative stress is an important reason for inducing autophagy.So we wondered to know whether AuNRs induce autophagy in A549 cells to be associated with oxidative stress.The results are as follows:1.Antioxidants MnTBAP and NAC were able to inhibit AuNRs-induced autophagyWestern blot results showed that the level of LC3-II expression in Con group was lower,which decreased slightly but not statistically significant after treatment with of MnTBAP and NAC.After treatment with AuNRs for 6 h,the expression of LC3-II was significantly increased,and this effect was reversed by MnTBAP and NAC.It is suggested that MnTBAP and NAC can ameliorate the autophagy induced by AuNRs in A549 cells.The results of laser scanning confocal microscopy showed that the expression of LC3 in Con group and NAC group was mainly expressed in the nucleus,indicating that the autophagy level was very low.After AuNRs treatment for 6h,the LC3 protein was transferred from the nucleus to the cytoplasm,and the expression of LC3 was gradually decreased and vacuolated.While the antioxidant NAC can reverse these effects.2.Antioxidants Mn TBAP and NAC were able to reverse the increase of ROS induced by AuNRsThe results of laser scanning confocal microscopy showed that after A549 cells were treated with different concentrations of AuNRs for 6h,ROS positive cells were not observed in Con group which indicating that ROS level was very low.With the increase concentration of AuNRs,the number of positive ROS cells and the green fluorescence were significantly increased in a dose-dependent manner,indicating that AuNRs could induce ROS production.The higher the concentration of AuNRs,the higher the level of ROS.While the antioxidants MnTBAP and NAC can reverse the above effects.3.Antioxidants MnTBAP and NAC were able to improve the oxidative stress induced by AuNRsThe results of T-AOC and GSH/GSSG showed that T-AOC,GSH/GSSG of the AuNRs treated group was significantly lower than that of the Con group,indicating that the antioxidant capacity of the cells treated with AuNRs was significantly decreased,and treatment of MnTBAP and NAC for 6h could reverse the above effects.4.Antioxidant NAC was able to reverse the decreased mitochondrial function induced by AuNRsMitochondrial membrane potential and ATP content results showed that the mitochondrial membrane potential and ATP content of AuNRs treated group were significantly lower than that of Con group,which indicated that the mitochondrial function of AuNRs was decreased after treatment with Au NRs,and treatment of NAC for 6h could reverse the above effects.Western blot results showed that UCP2 expression was significantly decreased in AuNRs treated group.The expression of UCP2 protein was significantly increased after treatment of NAC for 6h,which indicated that the change of mitochondrial function was related to the decrease of UCP2 protein expression.Conclusion:1.AuNRs can enter A549 cells,which can cause cell ultrastructural changes.AuNRs have a certain cytotoxicity in a dose and time-dependent manner.The higher of AuNRs concentration,the longer time of AuNRs exposure,the greater the cytotoxicity are present.2.Autophagy of A549 cells induced by AuNRs can be inhibited by autophagy inhibitor CQ.The expression of LC3,ATG16,ATG4 and Beclin-1 are increased and the expression of P62,a kind of autophagic substrate protein,is decreased in a dose-dependent manner.3.AuNRs can influence mitochondrial-related functions of A549 cells,then leading to reduced the ability of antioxidant stress and increased ROS levels,which mediates autophagy induced by AuNRs in A549 cells.