Axially Polarized O–Fe–N ₄ Single-Atom Enzyme Drives Clearance of Tumor-Resident Intracellular Microbiota and Potentiates Immunotherapy

Tumor immunotherapy is constrained by the low-immunogenicity and immune escape mechanisms of "cold" tumors, and the intratumoral microbiota can further exacerbate local immunosuppression. Fe single-atom nanozymes (Fe SAzymes) have recently emerged as a compelling approach for tumor therapy, owing to their superior catalytic activity and immune-regulating capabilities. This study presents the development of an efficient FeNC SAzyme with axial O-Fe-N₄ polarized centers anchored on a two-dimensional graphene substrate. These polar centers effectively reduce the energy barriers of NADPH-oxidase (NOX)-like, peroxidase (POD)-like, and glutathione-oxidase (GSHOx)-like reaction pathways, a mechanism validated through density functional theory (DFT) investigations. Further DFT analysis reveals that this polar structure significantly enhances both the charge density at the Fe center and the density of states in its <i>d</i>-orbitals, thereby improving adsorption capacity for reaction intermediates and electron coupling efficiency. After loading the NO-prodrug (SNAPi) and introducing RGD, the SNAPiFe@RGD complex was formed, achieving tumor selectivity and on-demand activation. Ultrasound radiation can promote NADPH oxidation and enhance reactive oxygen species (ROS) generation. ROS interacts with NO to generate highly reactive peroxynitrite (ONOO^-) species, thereby inducing lipid peroxidation and ferroptosis, accompanied by clearance of intratumoral microbiota, which subsequently reshapes the immune microenvironment. In a tumor model with bacterial colonization, SNAPiFe@RGD combined with ultrasound irradiation promotes dendritic cells (DCs) maturation and T cells infiltration, and synergizes with the αPD-L1 antibody to effectively inhibit the growth of contralateral tumors. Overall, the constructed axial-coordination FeNC SAzymes platform integrates multiple mechanisms, including intratumoral microbiota clearance, redox-cascade catalysis, ferroptosis induction, and immune checkpoint blockade, thereby achieving a precise and spatiotemporally controllable cancer immunotherapy strategy.