| Copper selenide nanoparticles(Cu2-xSe NPs)are copper sulfide nanomaterials composed of metallic elements copper and chalcogen selenium.They are widely used for optical diagnostic imaging and photothermal therapy(PTT)due to their strong near-infrared(NIR)optical absorption.With the continuous expansion using of Cu2-xSe NPs,their biosafety has received widespread attention in recent years.A variety of nanomaterials have been reported to penetrate the blood-brain barrier into the central nervous system and cause neurotoxicity,but whether Cu2-xSe NPs could enter the central nervous system and whether they have neurotoxicity and the mechanism of neurotoxicity remains unclear.The present investigation provides direct evidence that the toxicity of Cu2-xSe NPs can be specifically exploited to kill rat pheochromocytoma PC-12 cells(a cell line used as an in vitro model for brain neurons research)in dose-and time-dependent manners.These cytotoxicity events were accompanied by PC-12 cells mitochondrial damage,adenosine triphosphate(ATP)depletion,production of oxidizing species(including reactive oxygen species(ROS),malondialdehyde(MDA)and hydrogen peroxide(H2O2)),as well as reductions in antioxidant defenses systems(glutathione(GSH)and superoxide dismutase(SOD)).Moreover,our in vivo study also confirmed that Cu2-xSe NPs markedly induced neurotoxicity and oxidative stress damage in the striatum and hippocampal tissues of BALB/c mice.These findings suggest that Cu2-xSe NPs induce neurotoxicity in PC-12 cells and BALB/c mice via oxidative stress damage.Objective:In this study,we will investigate the toxic side effects of Cu2-xSe NPs on the nervous system by specifically exploiting to kill PC-12 cells and BALB/c mice,as well as the mechanism of neurotoxicity needs to be studied further,which will provide a toxicological experimental basis for their safer biological applications.Methods:1.Preparation and characterization of Cu2-xSe NPsThe PVP-stabilized Cu2-xSe NPs were prepared by one-step synthesis method as the literature.Transmission electron microscopy(TEM,JEM-2100,Japan)was used to observe the morphology of Cu2-xSe NPs.The average hydrodynamic radius and surface charge of the Cu2-xSe NPs in distilled water and physiological saline were analyzed using a Malvern laser particle size analyzer(NanoZS90,Malvern,UK)to investigate the particle size range and stability in different dispersion systems.The near-infrared(NIR)absorption was measured via a spectrophotometer(CARY5000,USA).The energy-dispersive X-ray(EDS,JEM-2100)was used to analyze the composition of the Cu2-xSe NPs.2.The cytotoxicity and mechanism study of Cu2-xSe NPs on PC-12 cellsWe first evaluated the effects of Cu2-xSe NPs on the cell morphology of PC-12 cells using an inverted microscope.Next,an MTT assay was used to investigate the changes of cell viability by different concentration of Cu2-xSe NPs.To quantitatively analyze the apoptotic rate of PC-12 cells treated with Cu2-xSe NPs,annexin V-FITC/PI staining and flow cytometry were employed.To determine the effects of Cu2-xSe NPs on mitochondria in PC-12 cells,TEM was used to observe the mitochondrial ultrastructural changes.The diminished mitochondrial membrane potential(??m)and the ATP depletion in the PC-12 cells were analyzed the effects on mitochondrial function using the kits.To investigate the relationship between oxidative damage and cytotoxicity,the intracellular levels of ROS,H2O2,GSH,SOD,and MDA were measured by using ROS,H2O2,GSH,SOD,and MDA determination kits,respectively.3.The neurotoxicity and mechanism study of Cu2-xSe NPs on BALB/c miceBALB/c mice were used to successfully construct the in vivo neurotoxicity study model.They were randomly divided into two groups,the control and Cu2-xSe NPs groups,and were euthanized at 20 days after administration.The striatum and hippocampal tissues of a mouse in each group were harvested,while the paraffin-embedded tissues were sectioned and analyzed by hematoxylin and eosin(H&E)staining.TUNEL and immunohistochemical caspase-3 staining to analyze the apoptosis of striatum and hippocampus of BALB/c mice.To investigate the relationship between oxidative damage and neurotoxicity,another mice striatum and hippocampal tissues were also lysed and the levels of H2O2,MDA,SOD and GSH were measured by using commercial kits according to the manufacturers' instructions.Results:1.The morphology of the prepared Cu2-xSe NPs were spherical,uniform,highly-dispersed particles examining by TEM.The hydrodynamic diameter and ?-potential in distilled water and physiological saline of the Cu2-xSe NPs were measured daily for a week No changes were found,indicating high dispersion and stability of the NPs.The absorption in the NIR range was analyzed from 650 to 1350 nm and the highest absorption of Cu2-xSe NPs was found at 1070 nm.Moreover,energy-dispersive X-ray spectroscopy(EDS)showed that the atomic ratio of the Cu2-xSe NPs was 1.3:1(X=0.7)2.The exposure of PC-12 cells to gradient concentration of Cu2-xSe NPs,which resulted in an increase in the number of round and neuritedeficient cells compared to the control group,which exhibited stretched neurites in the periphery.The MTT results revealed that PC-12 cells exposed to different concentrations of Cu2-xSe NPs for 12 h,24 h or 36 h resulted in a significant decrease in cell viability in dose-and time-dependent manners.As well as,the percentage of apoptotic cells markedly increased the percentage of apoptotic cells as the annexin V-FITC/PI staining and flow cytometry shown.Our TEM results,loss of MMP and ATP depletion,all of these results suggest that Cu2-xSe NPs penetrate the cell membrane and accumulated in the mitochondria,causing mitochondrial damage in PC-12 cells.Our results showed that the treatment of PC-12 cells with Cu2-xSe NPs resulted in a significant increase in the production of ROS,H2O2,and MDA in dose-dependent manners and a marked decrease in the levels of GSH and SOD.Taken together,these findings indicated that Cu2-xSe NPs induced mitochondrial dysfunction and oxidative stress leading to cytotoxicity in PC-12 cells3.Our results also revealed the neurotoxicity of Cu2-xSe NPs in vivo studies.As shown in the sections of striatum and hippocampal tissues,H&E staining of the Cu2-xSe NPs treatment group exhibited significant changes in pathology,with signs of necrosis,infiltration of inflammatory cells,and fibrosis,compared to BALB/c mice intravenously injected with physiological saline.The results showed that Cu2-xSe NPs could cause nerve tissue damage in striatum and hippocampus of BALB/c mice.Additionally,Cu2-xSe NPs treatment dramatically increased the numbers of TUNEL-positive cells and cleaved caspase-3 immunoreactivity,both indicative of apoptosis,in the striatum and hippocampal tissues.Consistent with our in vitro results,H2O2 and MDA levels were increased in BALB/c mice treated with Cu2-xSe NPs,while the SOD and GSH activities were significantly decreased compared to the control group Such findings suggest that Cu2-xSe NPs could cause damage in the central nervous system of BALB/c mice by oxidative stressConclusion:1.Cu2-xSe NPs penetrated the PC-12 cells membrane and accumulated in the mitochondria,inducing mitochondrial dysfunction and affecting the redox balance system in PC-12 cells.Next,Cu2-xSe NPs induced dose-and time-dependent cell death and apoptosis,and the cytotoxicity associated with oxidative stress in PC-12 cells2.BALB/c mice were intravenously injected with Cu2-xSe NPs,causing damage to the striatum and hippocampus,and Cu2-xSe NPs-mediated neurotoxicity in vivo is associated with damage from oxidative stress3.In summary,our study demonstrated the neurotoxicological effects of Cu2-xSe NPs and the potential mechanism involved in oxidative stress.Our research provided the toxicological experimental basis for safe application of Cu2-xSe NPs,and it is of significance. |
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