Study On Photothermal Killing And Autophagy Induction By Bismuth Selenide In A549 Cells

Objective:Bismuth selenide?Bi₂Se₃?nanomaterials with good dispersibility,high stability and excellent photothermal conversion ability were prepared and applied in photothermal therapy?PTT?,and investigated whether it could induce autophagy of lung cancer A549cells under near infrared reflection?NIR?light,and further explored the role of autophagy in photothermal killing of A549 cells.Methods:Bi₂Se₃ nanosheets modified with polyvinylpyrrolidone?PVP?were synthesized by a“green”solvothermal method,and characterized by transmission electron microscope?TEM?,dynamic light scattering?DLS?,Zeta potential and UV spectrophotometer.Its photothermal conversion ability and photostability were investigated by the photothermal conversion test.Taking the A549 cell line as the cell model,the toxicity of Bi₂Se₃ to A549 cells was detected by CCK-8 method,and its biocompatibility was investigated.The effect of Bi₂Se₃ on the vitality of A549 cells under the irradiation of 808 nm NIR light with a power density of 1.0 W/cm²was also detected by this method.The photothermal effect of Bi₂Se₃ under NIR light irradiation was determined by combining with the live-dead staining method,and the cell apoptosis of each group was detected by flow cytometry.And autophagy of A549 cells in each group was detected by monodansylcadaverin?MDC?staining.Western blot?WB?was used to detect the expression of cell autophagy marker protein LC3,combined with ImageJ software to analyze the autophagy ability.Immunoblotting technique was used to detect the expression of cleaved poly ADP-ribose polymerase?cleaved-PARP?as well as the phosphorylation levels of p38mitogen-activated protein kinases?p38?,stress-activated protein kinase/c-Jun N-terminal kinase?SAPK/JNK?and protein kinase B?Akt?of A549 cells in each group.Results:TEM characterization results showed that the prepared Bi₂Se₃ was a sheet-like structure with a size of about 50 nm,and the high-resolution TEM?HRTEM?image demonstrated that it had clear lattice fringes with a spacing of 0.21 nm.DLS and Zeta potential results showed that the average hydrated particle size of the Bi₂Se₃ nanosheets was about 56.2 nm,and the Zeta potential was-8.78 mV,possessed good dispersion stability.The results of photothermal conversion experiment demonstrated that the temperature rising rate and level of Bi₂Se₃ presented time and concentration dependent under the irradiation of 808 nm NIR light with a power density of 1.0 W/cm²,the heating rate and level of Bi₂Se₃ were the highest at 100?g/mL,and the temperature was as high as 66.3?,indicating a remarkable photothermal conversion capacity and excellent photostability.The cytotoxicity test results in vitro showed that Bi₂Se₃ was almost non-toxic to A549 cells in the range of 0¹00?g/mL,with cell survival rate remained above 90%,indicating its good biological safety.Compared with the control group,there was no significant difference on cell viability in the irradiation-only group,indicating that NIR light exposure alone had no effect on the survival rate of A549 cells.After the Bi₂Se₃co-incubated with the cells was irradiated with 808 nm NIR light at a power density of1.0 W/cm²,the survival rate of A549 cells decreased significantly,and the killing effect of Bi₂Se₃ on A549 cells was significantly enhanced at a concentration of 50?g/mL.The results of the live-dead staining and flow cytometry experiment revealed that the photothermal killing efficiency of the NIR-irradiated Bi₂Se₃ treatment group was significantly higher than that of the pure NIR-irradiated treatment and pure Bi₂Se₃treatment groups,which further proved the significant photothermal killing effect of Bi₂Se₃.And the results of flow cytometry experiment showed that the photothermal killing of A549 cells by Bi₂Se₃ was mainly achieved by inducing cell apoptosis.The results of MDC staining displayed that there was almost no autophagy in the A549 cells of each group in the absence of light.However,after the Bi₂Se₃ co-incubated with the cells was irradiated with 808 nm NIR light at a power density of 1.0 W/cm²,the autophagosomes of A549 cells increased,and dense green fluorescence appeared in the cytoplasm.The results of western blot indicated that under the condition of irradiating Bi₂Se₃ with NIR light,the band color of cell autophagy protein LC3?LC3-II,LC3-I?deepened gradually.Analyzed by ImageJ,the LC3-II/I ratio increased significantly in a concentration-dependent manner,reflecting an increased autophagy level under this condition,and with the increased expression of autophagy protein,the expression of apoptotic protein cleaved-PARP was also increased,the corresponding phosphorylation level of Akt was weakened,and the phosphorylation levels of p38 and JNK increased.Conclusion:Bi₂Se₃ nanosheets were successfully prepared by a“green”solvothermal method,which was simple and easy to operate.The prepared Bi₂Se₃ nanosheets had excellent photothermal conversion capability and good biocompatibility.Under the 808 nm NIR light irradiation with a power density of 1.0 W/cm²,it had significant photothermal killing effect on cells and mainly killed A549 cells by inducing apoptosis.The killing mechanism might be that irradiating Bi₂Se₃ with NIR light could induce cell autophagy,increase the expression of apoptosis-related protein cleaved-PARP,and activate p38,SAPK/JNK,PI3K/Akt cell stress-related signaling pathways.