Cascade Nano-Enzymatic Regulation Of Macrophage Phenotype And Applications In Immunotherapy

Macrophage plasticity and heterogeneity endows macrophage the interconversion capacity between the M1&M2 phenotypes.Based on this,body immunosuppressive state and immuno-active state could be reversed upon a variety of internal and external stimulus,which provides an efficient way of immunotherapy.Immunotherapy is an efficient approach against tumor,sepsis,atherosclerosis,obesity and other diseases.Catalytic nanomedicines specifically catalyze molecules in the lesion sites,which brings them incomparable properties over traditional drugs.However,the complex pathological microenvironment limits the catalytical curative effects of single enzymes.Cascade catalytic enzyme can interact with a variety of substrates to achieve complex catalytic reactions.Cascade amplification strategy is used to achieve higher catalytic activities.So,cascade enzyme can effectively improve the therapeutic effect of catalysis.In this paper,the concept of catalytic immunotherapy was proposed by combining the cascade catalytic nanoenzymes with immunotherapy.Herein,the work about the design and construction of the cascade catalytic nanoenzymes,the deep drive into mechanism of macrophage polarization and the application of immunotherapy were carried out.The details are as followed:（1）Based on the Redox characteristics of transition metal copper,a novel artificial enzyme was constructed.Cu_2-XTe NEs possess two kinds of enzyme-like activities,including glutathione oxidase（GSHOx）and POD,which catalyze GSH depletion and ROS generation,respectively.The defect-induced near-infrared-II（NIR-II）absorption endows Cu_2-xTe NEs with NIR-II light enhance enzymatic activities.The possible catalytical reaction mechanism of Cu_2-xTe NEs was proposed and Michaelis-Menten kinetics was calculated.The catalytic efficiency for GSHOx-mimicking and POD-mimicking activity of Cu_2-xTe NEs was77.1μm^-1 s^-1 and 1.9μm^-1 s^-1,respectively.（2）It has been recognized that tumor cells are abundant with GSH to protect them from ROS damage.Based on this,Cu_2-xTe NEs with GSHOx-and POD-mimicking activities were proposed to deplete intracellular GSH and generate ROS concurrently,which synergistically elevated intratumor oxidative stress.Cu_2-xTe NEs showed selective cytotoxicity towards tumor cells（>90%）with impaired mitochondrial function.and expose tumor associated antigens via immunogenic cell death.The generated oxidative stress as a stimulus activated the NF-κB signaling pathway to polarize macrophage to the M1 phenotype.（3）Encouraged by the outstanding enzyme-mimicking activity and immune activating properties of Cu_2-xTe NEs,we proposed the concept of catalytic immunotherapy.Cu_2-xTe NEs mediated catalysis andαPD-1mediated immune checkpoint inhibition was combined to evaluated the in vivo antitumor performance in bilateral tumor-bearing mice.Cu_2-xTe NEs successfully reverse immunosuppressive tumor microenvironment through elevating intratumor oxidative stress in situ.In this way,M1 macrophage polarization,dendritic cell maturation,infiltration and activation of T helper（T_h）cells and cytotoxic T lymphocytes（CTLs）in tumors was build up step by step.So,Cu_2-xTe NE based catalytic immunotherapy successfully induced an immunological memory effect to suppress tumor metastasis.（4）Due to the complex metabolism in cells,it is difficult for single enzyme to achieve effective catalytic regulation.Based on this,we constructed CaP@LOX@Pt cascade catalytic nanoenzymes.system by co-loading Pt of natural enzyme LOX and Pt nanoclusters with CAT-mimicking activities was co-immobilization by the co-precipitation method.LOX activity was completely maintained after loading,and it could efficiently catalyze the oxidation of lactic acid to pyruvate.Meanwhile,cytotoxic H₂O₂ was decomposed into non-toxic H₂O and O₂under the CAT-mimicking activities of Pt nanoclusters.CaP@LOX@Pt NEs targeted macrophage and be endocytosed into cytoplasm for an intracellular catalysis with little cytotoxicity.At the same time,generated pyruvate was flow into the TCA cycle,which up-regulated the level of oxidative phosphorylation.reduce lactate down-regulated glycolysis in vitro.Therefore,the catalytic metabolic regulation strategy was realized.（5）Based on the metabolic regulation of CaP@LOX@Pt NEs,the M2polarization reprogramming of macrophages was studied.Transcriptome analysis showed that CaP@LOX@Pt NEs genetically reprogram macrophage metabolism and polarization.CaP@LOX@Pt NEs were biocompatible and were applicable for in vivo catalysis.With the treatment of CaP@LOX@Pt NEs,CLP-induced sepsis mouse lived longer,and the inflammatory cytokine storm caused tissue damage is effectively protected.With the concept of cascade catalytic immunotherapy,CaP@LOX@Pt NEs take their anti-inflammatory effects within the macrophage polarization in the in vivo sepsis model.