Manganese-doped zinc oxide Nano-agonists activating the cGAS-STING pathway to potentiate anti-tumor immunotherapy and reprogram the immunosuppressive tumor microenvironment.

The cyclic GMP-AMP synthase - stimulator of interferon genes (cGAS-STING) innate immune pathway is an intriguing, but still challenging, target for cancer immunotherapy. The current small molecule STING agonists have several limitations, such as low membrane permeability, rapid enzymatic degradation, lack of tumor accumulation and dose-limiting systemic toxicity. In this work, we report the design and synthesis of the manganese-doped zinc oxide nanoparticles (Mn-ZnO NPs), as a dual-functional tumor microenvironment (TME) responsive nano-agonist platform and comprehensive biological validation thereof. The acidic TME (pH 6.5, 68.4 ± 3.6% Zn2+/Mn2+ release at 24 h) leads to accelerated degradation of the Mn-ZnO NPs (5 mol% Mn) compared with the physiological pH (pH 7.4, 18.4 ± 1.8% Zn2+ and 14.4 ± 1.4% Mn2+ release), which allows for tumor-selective release of Mn2+ and Zn2+ ions. Mn2+ ions are highly potent to sensitize the cGAS-STING axis, lowering the cGAS activation threshold for recognizing self-DNA, and triggering robust phosphorylation of STING (p-STING/STING: 4.84 ± 0.24-fold increase), activation of TBK1, and type-I interferon (IFN-β) production (8.24 ± 0.64-fold increase; p < 0.001) in 4 T1 murine breast cancer cells. At the same time, ZnO-associated oxidative stress and Mn-dependent radical chemistry increase intracellular reactive oxygen species (ROS) that lead to immunogenic cell death (ICD) as demonstrated by the exposure of calreticulin (CRT) on cell surfaces (3.84 ± 0.24-fold), the release of HMGB1 and extracellular ATP (eATP), which foster dendritic cell (DC) maturation and antigen cross-presentation. Intravenous administration of Mn-ZnO NP (5 mg Zn/kg, corresponding to approximately 0.22 mg Mn/kg for the 5 mol% formulation; days 1, 4, 7) significantly inhibited the growth of the 4 T1 tumor (tumor inhibition rate: 72.4 ± 4.4%), increased the infiltration of CD8+ T cells in the tumor (4.84 ± 0.48-fold), decreased the number of regulatory T cells (Tregs: -58.4%), and repolarized immunosuppressive M2 to immunostimulatory M1 macrophages (M1/M2 ratio: 3.84-fold increase) in syngeneic tumor-bearing BALB/c mice. Combination with anti-PD-1 antibody resulted in superior anti-tumor efficacy (tumor inhibition rate: 91.4 ± 2.4%) and a strong abscopal effect on distal untreated tumors (64.4 ± 4.4% inhibition) as well as superior tumor-free survival after rechallenge (100% at 45 days; p < 0.001 vs. control). Mn-ZnO NPs exhibited a good safety profile, showing no toxicity in the major organs. Overall, these results provide proof of the potential of Mn-ZnO nano-agonists as a preclinical innate immune-activating platform that can convert immunologically cold tumors to immunologically hot tumors and that can synergize with checkpoint blockade immunotherapy.