Generation Of Nitric Oxide Via Cascade Raction In Artificial Cells For Blood Anticoagulation

Coacervate microdroplets,as a kind of artificial cell model,are compact droplets formed by biomolecules based on liquid-liquid phase separation.They have properties such as selective molecular enrichment,liquid fluidity,and controllable response assembly.As a cell model,coacervate microdroplets are widely used in the construction of biological carriers and microreactors.Nitric oxide(NO)is a signal molecule in the living body,which has important physiological regulation functions and is closely related to blood coagulation,vasodilation,antibacterial,and tumors.Since the half-life of NO is extremely short,it is difficult to achieve controlled release,and its physiological activity depends on the concentration level.Therefore,how to achieve the controllable production and release of NO is an important challenge for NO biomedical applications.Aiming at the problem of the controllable production and release of NO,this paper fixes the cascade enzyme reaction of NO production in the coacervate microdroplets,NO production system based on the coacervate microdroplet artificial cells.Two aspects of research work has been carried out as below:1.Spatially controlled cascade enzymatic production of NO in coacervate vesicles.Mixture of the positive and negative charged polyelectrolytes led to the formation of the molecular crowding coacervate microdroplets via liquid-liquid phase separation,and the surface was coated with a phospholipid membrane,which resulted in the preparation of coacervate vesicles(Coac Ves).The coacervate vesicles as an artificial cell,different biological enzymes(glucose oxidase GOx,horseradish peroxidase HRP and catalase CAT)were immobilized inside the coacervate vesicles and on the phospholipid membrane,and four different coacervate vesicles were obtained.Enzyme spatially distributed coacervate vesicles includes GOx/HRP@CAT,GOx/CAT@HRP,HRP/CAT@GOx and GOx/HRP.Glucose is oxidized by GOx to generate gluconic acid and H₂O₂.H₂O₂ and hydroxyurea are catalyzed by HRP to generate NO.At the same time,the intermediate H₂O₂ is catalyzed and decomposed into H₂O and O₂ by CAT.Taking coacervate vesicles as the microreactor,under the condition that the substrates are both glucose and hydroxyurea,the different spatial distribution of cascade enzymes on the coacervate vesicles leads to significant differences in output products.GOx/HRP@CAT only produces NO;GOx/CAT@HRP does not pr oduce NO and H₂O₂;HRP/CAT@GOx only produces H₂O₂;and GOx/HRP simultaneously produces H₂O₂and NO.In this work,four different coacervate vesicles were developed and the final products were modulated through different spatial layouts of cascade enzymes.2.Generation of nitric oxide in coacervate vesicles and its anticoagulant applications.Four different coacervate vesicles with different spatial distribution of enzyme cascade were designed.GOx/HRP@CAT coacervate vesicles were prepared by encapsulation of GOx/HRP in the coacervate microdroplets,and immobilization of CAT on the phospholipid membrane.The coacervate vesicle s were used as artificial cells for NO production,and 5?M NO can be produced within 2 h,and the intermediate product H₂O₂,which is unfavorable to cells,is decomposed by CAT filtration on the phospholipid membrane.This system is used in anticoagulation research.NO can inhibit the activation,adhesion and aggregation of platelets and the generation and activation of thrombin,a key component of the coagulation process.Therefore,the coacervate vesicle cascade enzyme avoids the overflow of intermediate H ₂O₂,and can control the production and release of NO,which provides an effective guarantee for the further biomedical applications of NO.