Preliminary Exploration Of The Death Mechanism Of Head And Neck Squamous Cell Carcinoma Cells Induced By Photothermal Effect Of Gold Nanorods Modified By PD-L1 Monoclonal Antibody

Objectives:Gold nanorods generate surface plasmon resonance under the irradiation of near-infrared light and release corresponding heat at the same time,which can induce tumor cell death,which is a physical hyperthermia mode.The method of cell death strongly depends on the magnitude of the thermal stimulus load and shows a complex intermediate form or the coexistence of multiple death modes from active programmed death(such as apoptosis,cell senescence)to passive death(such as necrosis).Our team’s previous research found that gold nanorods can be modified by monoclonal antibodies to achieve tumor targeting,and can induce programmed death of head and neck cancer cells under appropriate NIR laser irradiation.However,the specific mechanism is not yet clear.Therefore,this topic will further explain the PD-L1 modified gold nanorods inducing the senescence of head and neck squamous cell carcinoma cells to induce the programmed death mechanism of head and neck squamous cell carcinoma,for the subsequent treatment of head and neck squamous cell carcinoma with gold nanorods.Provide theoretical basis.Methods:1.Preparation of gold nanorod conjugated PD-L1 antibody.2.Detect the maximum light absorption peak wavelength of PD-L1mAb/AuNRs and the coupling rate of PD-L1mAb by UV-visible spectrophotometer.3.PD-L1mAb/AuNRs was analyzed by EDS for its trace elements.4.Zeta potential characterizes the physical stability of PD-L1mAb/AuNRs.5.CCK-8 method was used to detect the effect of PD-L1mAb/AuNRs on cell proliferation of head and neck squamous cell carcinoma cells.6.SA-β-Gal staining to observe the senescence of head and neck squamous cell carcinoma cells after PD-LlmAb/AuNRs photothermal effect.7.CCK8 detects the effect of 24h,48h,72h cell viability of Hep-2 cells after PD-L1mAb/AuNRs photothermal treatment.8.Flow cytometric detection of apoptosis in Hep-2 cells 24h,48h,72h aflter PD-L1mAb/AuNRs photothermal treatment.9.SA-β-Gal staining to observe the senescence of Hep-2 cells 24h,48h,72h after PD-L1mAb/AuNRs photothermal treatment.10.Immunofluorescence detection of the expression of DNA damage markers y-H2AX and 8-OH-dG 24h,48h,72h after photothermal treatment of PD-L1mAb/AuNRs on Hep-2 cells.11.Western blot detection of PD-L1mAb/AuNRs photothermal treatment of Hep-2 cells 24h,48h,72h senescence-related protein P16,P21,CDK2 protein expression.Results:1.The maximum absorption wavelength of PD-L1mAb/AuNRs longitudinal band is 783 nm,which can have the maximum penetration depth in biological tissues.2.The coupling rate of PD-L1mAb to AuNRs is 34%.3.PD-L1mAb/AuNRs has good physical stability and is suitable for biomedical applications.4.Hep-2 cells and CNE-2 cells were treated with PD-L1mAb/AuNRs,6h after NIR irradiation,the cell viability decreased significantly,and the effect on the viability of NP69 cells was not significant.5.The photothermal effect of PD-L1mAb/AuNRs can induce senescence in Hep-2 cells and CNE-2 cells.6.The photothermal effect of PD-L1mAb/AuNRs can decrease the cell viability of Hep-2 cells,and after the laser irradiation is finished,the cell viability still shows a downward trend with the increase of time within a certain time range.7.The photothermal effect of PD-L1mAb/AuNRs can cause obvious apoptosis of Hep-2 cells,and after the laser irradiation,the apoptosis rate still shows an upward trend with the increase of time within a certain time range.8.PD-L1mAb/AuNRs can inhibit the DNA replication of Hep-2 cells under NIR irradiation,so that most of the cell cycle stops in the G1 phase.9.The photothermal effect of PD-L1mAb/AuNRs can significantly increase the number of Hep-2 cell senescence,and after the laser irradiation,the number of cell senescence still shows an upward trend with the increase of time within a certain time range.10.After the photothermal effect of PD-L1mAb/AuNRs,the expression of DNA damage markers y-H2AX and 8-OH-dG in Hep-2 cells can be observed to increase significantly,and after the end of NIR irradiation,the increase over time within a certain time range The expression of DNA damage markers y-H2AX and 8-OH-dG is still showing an upward trend,indicating that Hep-2 cell DNA is damaged,increasing the degree and number of cell aging.11.After the photothermal effect of PD-L1mAb/AuNRs in Hep-2 cells,the positive gene protein P21 and P16 expression in the DNA damage response pathway were up-regulated,and the negative gene protein CDK2 expression was down-regulated.Increased,the expression of P16 and P21 showed an upward trend;the expression of CDK2 showed a downward trend.Conclusions:1.The photothermal effect of PD-L1mAb/AuNRs can effectively kill head and neck squamous cells.2.The photothermal effect of PD-1mAb/AuNRs can induce necrosis,apoptosis and senescence of head and neck squamous cell carcinoma cells.3.The photothermal effect of PD-1mAb/AuNRs may induce Hep2 cell senescence through the p16INK4a/Rb signal pathway and the p53/p21Cip1 signal pathway.