PH-Responsive Motion And Applications Of Organic-Inorganic Dual-Enzyme Cascade-Driven Nanomotors

Biological enzymes are a kind of proteins with high catalytic efficiencies and substrate catalytic characteristics.Micro-nanomotors,driven by biological enzymes,can release chemical energy stored in fuel molecules（glucose,urea,etc.）into their own mechanical kinetic energy through gentle enzymatic reactions,resulting in autonomous movement.Although there are a large number of biological enzymes and their corresponding substrates in nature,the most studied nanomotors driven by enzymes are mainly based on glucose oxidase,catalase and urease.Compared with the other two,the micro-nanomotor driven by glucose oxidase has the following advantages:First,fuel of glucose as an essential energy supply substance for human cell metabolism,has excellent biocompatibility,and can achieve the purpose of independent regulation of motor movement behavior through exogenous intake.Second,glucose oxidase is often used in a cascade reaction with other enzymes to boost motor power while catalyzing hydrogen peroxide,a toxic byproduct.However,currently developed glucose oxidase-driven nanomotors still have the following problems,which limit their application in the field of biomedicine:First,insufficient driving force leads to low motor speed.Currently developed glucose oxidase drives the nanomotor.Due to the pore structure of the carrier,GOx can only be fixed on the surface of the carrier,resulting in a low enzyme content per unit volume of the motor,which greatly limits the catalytic efficiency of GOx to a certain extent,and thus reduces the movement rate of the micro-nanomotor.Second,the size of the micromotor driven by biological enzymes currently developed is mostly concentrated in（sub-）micron level,which is easy to cause vascular embolism and other life-threatening diseases after entering the human body,so it is not conducive to the application in the field of biomedicine.Third,the function of the motor is single and the degree of intelligence is insufficient.Currently,the micronanomotors driven by GOx cascade reactions are mostly constructed by organic enzymes（such as catalase）,and their purpose is limited to consumption of H₂O₂ as a by-product to improve biocompatibility.In order to solve the above problems,we developed an intelligent responsive nanomotor driven by a cascade reaction using glucose as"fuel".The main research content includes two parts:First,anion-assisted liquid phase self-assembly was used.Triethylamine（TEA）was used as the catalyst,TEOS was used as the silicon source,and the cationic surfactant cetyltrimethylammonium bromide（CTAB）and anionic sodium salicylate（Na Sal）as the structural guiding agent.Dendritic mesoporous silica carrier DMSNs was prepared.The influence of the ratio of saline to CTAB on the pore parameters of the carrier was investigated.With the CTAB/Na Sal=1,the DMSN-1 was highly dispersed,with a large pore size distribution（32 nm）,a large specific surface area（435.34 m²/g）,and a good perforation with a small particle size（200 nm）.The effect of pore structure and enzyme active site on enzyme immobilization was also studied.Then d MSN-1 was used as the immobilized carrier and the maximum amount of GOx was obtained by controlling the addition of APTES at 0.52 mmol（G_L=255±10 mg/g）.Then the optimal immobilized enzyme activity could be obtained by adjusting the immobilized time and the initial concentration of the enzyme to 12 h and 3 mg/m L,respectively.Secondly,on the basis of the above work,the inorganic nano-enzyme Fe₃O₄ NPs was further loaded,and the janus structure was constructed by magnetron sputtering.Finally,the GOx-Fe₃O₄-JNDMSN nanomotor driven by the bioenzyme-inorganic nano-enzyme cascade reaction with glucose as fuel was successfully prepared.The prepared nanomotor has good dispersibility and the particle size distribution is about240 nm.Then,the GOx-Fe₃O₄ cascade reaction products·OH and O₂ were detected in different pH glucose aqueous solutions,which verified the successful construction of the intermediate reaction of nanomomotor.In addition,the maximum movement rate of the nanomotor is 2.07±0.48μm/s in a glucose solution of 200 m M,which proves the feasibility of the cascade reaction to drive the nanomotor efficiently.Then,by changing the pH value of glucose aqueous solution,when the pH value increases from 3 to 7.4,the motion rate also reaches the maximum of 2.14±0.31μm/s,which proves the pH responsive motion behavior of nanomoters.Finally,the cytotoxicity test results of GOx-Fe₃O₄-JNDMSN nanomotor showed that under acidic conditions（pH=6.5）,the inhibitory rate of GOx-Fe₃O₄-JNDMSN nanomotor on the survival rate of MCF-7human breast cancer cells was over 65%.In this work,the intelligent responsive motion behavior of nanomotor driven by glucose cascade reaction is proposed,which is expected to provide new insights for the application of nanomotor in the biomedical field.