Preparation Of Cerium Oxide-modified Gold Nanorods And Their Application In Photoimmunotherapy For Triple Negative Breast Cancer

Objective:Triple-negative breast cancer（TNBC）was closely associated with high metastatic risk and mortality and has not yet found a targeted receptor for targeted therapy.Cancer immunotherapy especially photoimmunotherapy shows promising potential in TNBC treatment because of great spatiotemporal controllability and non-trauma.However,the therapeutic effectiveness was limited by insufficient tumor antigen generation and the immunosuppressive microenvironment.Herein,we prepared cerium oxide（Ce O₂）end-deposited gold nanorods（CEG NRs）to achieve excellent near-infrared photoimmunotherapy and fluorescence imaging.Followed by modification with Cy7,CEG-Cy NRs can emit fluorescence signal and be used as fluorescence imaging agent for tumor diagnosis.CEG NRs can efficiently generate hot electrons and avoid hot-electron recombination to release heat and form reactive oxygen species（ROS）,showing strong photothermal and photodynamic effects to destroy tumors,triggering immunogenic cell death（ICD）and activating part of the immune response.Simultaneously,combining with anti-PD-1 antibody could further reverse the immunosuppressive microenvironment and thoroughly activate the immune response by enhancing cytotoxic T lymphocytes infiltration,thus achieving excellent photoimmunotherapy.Methods:GNRs were synthesized by seed mediated-method,and then modified with Ce O₂ for further surface growth into CEG NRs or CBG NRs.The physical properties were tested via transmission electron microscopy（TEM）and X-ray diffraction（XRD）.The photothermal properties of CEG NRs were measured by infrared thermal imager.DCF,as the ROS indicator,was used to evaluate the ability of CEG NRs to produce ROS under 808 nm near-infrared laser irradiation.Murine breast cancer 4T1 cells were incubated with CEG NRs for24 h to evaluate the biosafety of CEG NRs in vitro.DCFH-DA probe was used to evaluate the ROS production level of CEG NRs after co-incubation with cells.The mechanism of cell damage induced by CEG NRs during ROS production was studied by western blot.Flow cytometry was used to investigate the apoptosis induced by CEG NRs after co-incubation with cells.Mouse breast cancer 4T1 cells were subcutaneously inoculated into the back of BALB/c female mice to establish a mouse tumor model.After intravenous injection of CEG-Cy7 NRs,fluorescence live imaging was performed to evaluate its performance as a fluorescence contrast agent.The therapeutic effect and biosafety of CEG NRs were evaluated by studying body weight changes,tumor volume,and histochemical analysis of tumors and organs in mice.Results:The structure,chemical formation and element distribution of CEG NRs were observed by TEM image and XRD pattern.The temperature-rise curve of CEG NRs indicates that they has good photothermal conversion ability.DCF fluorescence test proved that CEG NRs had a strong ability to produce ROS.CEG NRs showed no obvious cytotoxicity without 808nm laser irradiation,which indicated that CEG NRs had good biocompatibility.With 808nm laser irradiation,CEG NRs could kill tumor cells in a dose-dependent manner.Western blot analysis showed that CEG NRs up-regulated the expression of HSP-70 and HO-1 in a dose-dependent manner.Flow cytometry showed that CEG NRs could induce a large number of apoptosis and destroy tumor cells under NIR laser irradiation.Fluorescence images of tumor-bearing mice showed that CEG-Cy7 NRs could aggregate in tumor areas and emit obvious fluorescence signals.Combined with photoimmunotherapy and PD-1 blocking therapy,CEG NRs has a significant inhibitory effect on tumor tissues and is biosafe.Conclusion:In this study,GNRs were synthesized by seed-mediated method,followed by further growth of Ce O₂ into CEG or CBG NRs by surface modification to achieve excellent NIR photoimmunotherapy.Compared with CBG NRs,the LSPR of CEG can be regulated to the NIR region,possessing strong photothermal conversion ability and ROS generation under NIR light irradiation,resulting from the spatial distribution structure that makes hot electrons participate at the ends and hot holes be consumed at the exposed side surface,greatly enhancing the utilization efficiency of photocarriers,thus showing excellent photothermal and photodynamic effects to destroy tumors and activate part of the immune response.Simultaneously,combining with PD-1 antibody could reverse the immunosuppressive microenvironment and thoroughly activate the immune response by enhancing cytotoxic T lymphocytes infiltration.In addition,CEG-Cy7 NRs accumulated at the tumor site and only at the tumor site at 24 h,which can be used for tumor fluorescence imaging in mice to observe the tumor location and morphology.In conclusion,this study demonstrates the superiority of photoimmunotherapy combined with PD-1 blocking therapy and fluorescence imaging in the integration of diagnosis and treatment of TNBC.