Mace-like Plasmonic Au Pd Heterostructures Boost Near-Infrared Photoimmunotherapy

Objective:In recent years,photoimmunotherapy has provided a new targeted therapy strategy for highly malignant triple negative breast cancer（TNBC）due to its spatiotemporal precision and non-invasive characteristics.However,their therapeutic effect is severely restricted by the insufficient generation of tumor antigens and the weak activation of immune response,which was caused by the limited tissue penetration of light and complex immunosuppressive microenvironment.Therefore,there is an urgent need to develop a novel photoimmune agent that can produce sufficient tumor antigens and effectively activate innate immunity for primary and metastatic TNBC therapy.In this study,we have fabricated mace-like plasmonic Au-Pd heterostructures（Au Pd HSs）to boost near-infrared（NIR）photoimmunotherapy.（1）Firstly,the photothermal and photodynamic effects of Au Pd HSs under near infrared light were investigated to see whether it could effectively trigger immunogenic cell death（ICD）and activate the immune response.（2）The mechanism of action of the tip surface of Au Pd HSs on DCs.（3）Finally,the effect of Au Pd HSs combined with anti-programmed death ligand 1（α-PD-L1）on primary and metastatic TNBC was explored.This study provides a new scheme and theoretical basis for TNBC immunotherapy combined with nanotopological structure and plasma properties.Methods:This study was conducted in three parts:physical characterization,photothermal photodynamics,cell experiment and animal experiment of Au Pd HSs.（1）Physical characterization of Au Pd HSs:Au nanorods were synthesized by seed-mediated growth method and used as seed solution.By controlling the amount of Na Pd Cl₄,the plasma resonance absorption peak of Au nanorods was adjusted to close to 800 nm,and then the spiky plasma Au Pd nanoheterogeneous materials were obtained.The materials were analyzed by transmission electron microscope（TEM）,high resolution transmission electron microscope（HRTEM）,scanning transmission electron microscope（STEM）images,X-ray absorption spectrum（X-ray）,X-ray diffraction（XRD）,hydration particle size and Zeta potential,UV-Vis-near-infrared absorption spectrum（UV-Vis-NIR）.In order to improve the biocompatibility of Au Pd HSs,the surface of Au Pd HSs was combined with the mercaptoterminal PEG（PEG-sh,Mw=5000）to obtain peg-modified Au Pd HSs（pAu Pd HSs）.The photothermal properties of Au Pd HSs were measured by infrared thermal imager.Total reactive oxygen species（ROS）,hydroxyl radical（·OH）,monomer oxygen（¹O₂）and superoxide anion（O₂·^-）were evaluated by DCF,APF,SOSG and superoxide anion kits at different concentrations of Au Pd HSs（25,50,100μg/m L）.（2）Cell experiment:Mouse breast cancer 4T1 cells were co-incubated with pAu Pd HSs labeled with Rh B,and confocal fluorescence microscopy（CLSM）was used to analyze and evaluate the cells’enucleation ability to nanomaterials.The in vitro phototherapy effect of pAu Pd HSs（0～100μg/m L）was measured by CCK-8 method.The PTT and PDT effects of pAu Pd HSs were evaluated by calcein acetoxymethyl（AM）/propyl iodide（PI）staining in living/dead cells.Intracellular ROS levels were evaluated by DCFH-DA probe after pAu Pd HSs treatment.The mechanism of ROS production by pAu Pd HSs induced cell damage was confirmed by Western Blot.The expression of DCs costimulatory molecules（CD80,CD86）was analyzed by flow cytometry to study the in vitro immunostimulative ability of pAu Pd HSs on DCs.The efflux of K⁺was detected by sensitive fluorophore fluorescence probe（PBI-AM）.The in vitro immunostimulation of DCs with phototherapy was confirmed by Transwell system.The levels of IL-1βand Caspase-1,biomarkers of inflammasome,were detected by ELISA and western blot.Apoptosis was determined by flow cytometry.（3）Animal experiments:A 4T1 tumor xenograft model was designed and mice were divided into nine groups to explore the anti-tumor effect of pAu Pd HSs in vivo.The biological distribution of pAu Pd HSs in mice was tracked by fluorescence imaging and inductively coupled plasma emission spectrometry（ICP-OES）.The in vivo PTT effect of pAu Pd HSs was evaluated by monitoring the temperature variation of tumor area under NIR irradiation with mouse infrared thermal imager.Tumor growth curves and actual tumor size were recorded for both in situ and distal metastases.H&E and TUNEL staining were used for pathological analysis of tumor sections.The maturation of DCs in tumor tissues and lymph nodes was evaluated by flow cytometry,and the contents of CD8⁺and CD4⁺T cells in CD3⁺T cells in tumor tissues were analyzed.Results:（1）TEM observation showed that the prepared Au Pd HSs had a monodisperse hammer nanometer morphology.Based on the images,the length of Au NRs was 45.9±7.7 nm and the diameter was 10.9±3.5 nm.The Pd tip was evenly wrapped on the inner nucleus of Au NRs.The average length was 6.1±1.9 nm;HRTEM images showed that the interfacial distance of 0.2 nm corresponded to the（200）surface of Au NRs nucleus,and 0.19 nm and0.22 nm corresponded to the（200）and（111）lattice surface of Pd,respectively;The structure and element distribution of Au Pd HSs were confirmed by STEM images and X-ray.XRD showed the diffraction peaks of Au and Pd.UV-Vis-NIR showed that the LSPR peak of Au NRs was redshifted from 730 nm to 810 nm by the preparation of Au Pd HSs.The hydrodynamic size of Au Pd HSs increased from 141±8.3 nm to 163.7±11.7 nm;After the solution storage for 6 months,the absorption profile,hydrodynamic size and zeta potential of pAu Pd HSs remained unchanged,indicating that pAu Pd HSs had good stability;The temperature rise curve of pAu Pd HSs under NIR laser irradiation shows that the synthesized material has excellent photothermal conversion ability,which is concentration-dependent and power-density-dependent.The results of the production of total ROS,·OH,¹O₂ and O₂·^-in vitro showed that the production of these reactive oxygen species was concentration-dependent,which confirmed that pAu Pd HSs had good photothermal photodynamic properties.（2）CLSM analysis showed that 4T1 cells had obvious intake of pAu Pd HSs;The results of CCK-8 method show that the nanoparticles have good biocompatibility.Western blot showed that pAu Pd HSs could significantly up-regulate the expression of HSP70 and HO-1 under NIR laser irradiation,thus activating oxidative stress signaling pathway,leading to ROS production and mitochondrial dysfunction.In cell experiments,it was confirmed that pAu Pd HSs exerted photothermo-photodynamic effect to produce ROS and eventually induce cell death under NIR irradiation;The results of costimulatory molecules CD80 and CD86 of DCs by flow staining showed that the spiny pAu Pd HSs nanomaterials could increase the proportion of mature DCs by 20%.It was further verified that DCs maturation was related to K⁺efflux and activation of inflammatory bodies.The results of PBI-AM probe indicated that pAu Pd HSs could significantly inhibit K⁺efflux.ELISA and Western blot results showed that pAu Pd HSs could significantly improve IL-1βlevel and Caspase-1expression,which confirmed that the tip surface of pAu Pd HSs could exert mechanical stress,cause K⁺effusion,activate inflammatory bodies of DCs,and further mature DCs.To promote antigen presentation.The transwell co-culture system mimicking in vivo tumor ablation confirmed that tumor-associated antigens produced by 4T1 cells or fragments under NIR irradiation promoted DCs maturation.The above results demonstrated that the combination of pAu Pd HSs plasma nanomaterials and phototherapy can significantly enhance the immune response of DCs and ultimately induce anti-tumor immune response.（3）In vivo animal experiments showed that pAu Pd HSs accumulated significantly in tumor 24 h after injection in vivo,which proved that pAu Pd HSs could be passively targeted to tumor tissue.Infrared thermal imaging results showed that the tumor temperature increased from 25.1°C to about 42.8°C under NIR laser irradiation after caudal vein administration;The tumor growth curve and actual tumor size indicated that pAu Pd HSs combined withα-PD-L1 could effectively inhibit bilateral tumor growth under NIR,and H&E and TUNEL staining were consistent with the above results.The proportion of mature DCs in tumor tissues and lymph nodes analyzed by flow cytometry was significantly higher than that in all other groups.The contents of CD8⁺and CD4⁺T cells in CD3⁺T cells indicate that pAu Pd HSs combined withα-PD-L1 treatment under the action of NIR has a significant anti-tumor immune response effect.Conclusion:Hammer plasma Au Pd HSs was synthesized to enhance near infrared immunotherapy.The Pd tip is evenly wrapped on the Au NRs,which not only successfully regulates the LSPR to the near infrared region,but also greatly promotes the production and transfer of hot electrons,showing excellent near infrared PTT and PDT.Meanwhile,the spiny surface of Au Pd HSs can promote DCs maturation and activation,and enhance immune response.In addition,Au Pd HSs in combination with clinically approvedα-PD-L1 can effectively relieve immune tolerance and promote intratumoral invasion of CTLs.Finally,CTLs promote the secretion of cytokines and enhance cancer immunotherapy.In conclusion,Au-Pd HSs has a good immunoactivation effect during the elimination of in situ and distal tumors.This work shows great potential for the combination of physical immune activation and photoimmunotherapy,providing a new strategy for the clinical treatment of TNBC.