A polymeric triple-enzyme nanoparticle by light activatable crosslinking for cascade catalytic therapy of osteosarcoma

Multi-enzymatic cascade reactions play fundamental roles in biosynthesis, energetic metabolism, cell signaling, proliferation and apoptosis in living cells. Such reactions have been proposed for cancer therapy via overproducing reactive oxygen species, depleting nutrients for tumor starvation, or remodeling immunosuppressive microenvironment, however, co-delivery of multiple enzymes with optimal cascade catalytic activity remains challenging. Here, we propose a polymer nanoparticle loaded with glucose oxidase (GOx), lactate oxidase (LOx), and chloroperoxidase (CPO) for synergistic cancer catalytic therapy. The enzymes were complexed with O-nitrobenzyl-modified ε-polylysine via ionic and hydrophobic interactions, followed by light-activated crosslinking to prepare serum-stable and pH-responsive protein nanoparticles. The GOx and LOx in the nanoparticle efficiently deplete glucose and lactate, generating excessive hydrogen peroxide, while CPO further catalyze hydrogen peroxide in the presence of chloride ions to produce singlet oxygen (¹O₂), promoting both apoptosis and ferroptosis in cancer cells. The triple-enzyme polymer nanoparticles efficiently inhibit tumor growth in an osteosarcoma model without causing obvious adverse effects. This multi-enzyme delivery system provides a promising tool for the treatment of diseases via multi-enzymatic cascade reactions.