Construction Of Magnetic-Responsive Enzymenanozyme Cascade System And The Mechanism Of Activity Regulation

Multi-enzyme catalysis is widely used in biomedical,environmental protection,and industrial production.Modulating the kinetic matching between enzymes is crucial for efficient cascade catalysis.The nanomaterials-based multi-enzyme systems can perform the initial cascade reaction and respond to exogenous physicochemical stimuli,which will provide the possibility for the spatiotemporal control of the multi-enzyme systems.However,how to precisely modulate the kinetic matching between enzyme-nanozyme at the molecular level remains a challenge.Magnetic nanomaterials not only have the catalytic properties of nanozyme but also can convert the energy from alternating magnetic fields（AMF）into nanoscale heat,which subsequently regulate nano-cascade enzymes catalysis.Therefore,we choose glucose oxidase（GOx）and ferrimagnetic vortex iron oxide nanoring（Fe₃O₄NR）as a model enzyme-nanozyme cascade system and fabricate a series of magnetic-responsive Fe₃O₄NR@GOx with nanoscale inter-enzyme distances.The catalytic performance,magneto-heat modulation mechanism,and antitumor effect of this cascade enzyme system have further been investigated.The key findings are as follows:（1）Fe₃O₄NR@GOx nano-cascade enzymes construction and magneto-heat properties,nanozyme activity characterization:Fe₃O₄NR（64 nm）prepared by hydrothermal method,was subsequently functionalized using polyethylene glycol molecules of different chain lengths.Fe₃O₄NR and GOx were covalently linked via EDC/NHS catalysis.Four Fe₃O₄NR（D0）/（D2）/（D5）/（D10）@GOx nano-cascade enzymes with varying subnanometer linking distances were successfully constructed.The magneto-heat performances test showed that the specific absorption rate（SAR）value of Fe₃O₄NR was 12 times higher than that of superparamagnetic Fe₃O₄ nanoparticles.Meanwhile,the enzyme activity assay demonstrated that Fe₃O₄ NR exhibited intrinsic peroxidase-like catalytic activity.（2）Distance-dependent catalytic performance of Fe₃O₄NR@GOx nano-cascade enzymes:The comparison of the natural cascade enzyme and the nano-cascade enzyme systems revealed that the mismatch between the kinetics of Fe₃O₄NR and GOx is intrinsically responsible for the poor efficiency of the cascade reaction.The changes in the cascade reaction rate and the intermediate H₂O₂ indicated that the distance-optimized Fe₃O₄NR@GOx nano-cascade enzyme could promote the Fe₃O₄nanozyme activity.It further enhances both the kinetic matching between Fe₃O₄NR and GOx,and the cascade reaction efficiency.Compared to the free GOx&Fe₃O₄NR mixture,four Fe₃O₄NR@GOx nano-cascade enzymes appeared a significant increase in the cascade activity by 34–332 times,and particularly the Fe₃O₄NR（D2）@GOx exhibited the highest catalytic activity.（3）Modulation of Fe₃O₄NR@GOx cascade reaction kinetics by magnetic field actuation:The Fe₃O₄NR serves as a local inductive heat source under AMF to trigger activity changes of GOx and Fe₃O₄ nanozyme in a distance-dependent manner,thereby modulating the cascade reaction kinetics of various Fe₃O₄NR@GOx.A larger activity enhancement on the rate-limiting step is shown to greatly improve the kinetics match between GOx and Fe₃O₄nanozyme,leading to more efficient intermediate H₂O₂ utilization and more prominent cascade activity enhancement.Fe₃O₄NR（D2）@GOx under AMF exhibits higher cascade activity than the free GOx&Fe₃O₄ NR mixture over 400-fold..（4）In vitro and in vivo antitumor performance of Fe₃O₄NR@GOx nano-cascade enzymes by magnetic field actuation:AMF-triggered Fe₃O₄NR@GOx nano-cascade enzymes induced more ROS production,which further improved the killing effect on tumor cells.In vivo antitumor experiments demonstrated that AMF-treated Fe₃O₄NR@GOx exhibited a more powerful tumor suppression effect on 4T1 tumor-bearing mice.These Fe₃O₄NR@GOx combined with AMF displayed similar patterns of in vivo antitumor effect and in vitro catalytic activity.Meanwhile they also exhibited good biocompatibility and held potential for clinical application.In summary,this work presents a strategy to construct a magnetic-responsive natural enzyme-nanozyme cascade systems based on distance optimization,and the kinetic process of the cascade reaction was precisely regulated by the magneto-heat effect to realize the efficient tumor treatment.The findings may provide valuable insights into the design of multi-enzyme systems and technical supports for the precise regulation of more cascade reactions in the future.