Biological Membrane-enclosed Coacervate Microdroplets For Nitric Oxide Generation And Application

Nitric oxide（NO）is a gas molecule and is associated with many physiological and pathological processes such as vasodilatation,anticoagulation,antibacterial and tumor elimination.Currently,NO donor compounds and NO delivery vehicles have received wide attentions in biomedicine.It is a great challenge to modulate the production and release of NO in a controlled manner due to its short half-life,low stability,and concentration-dependent functions.Coacervate microdroplets are formed by a liquid-liquid phase separation that are driven by an initial electrostatic attraction between oppositely charged macroions.Coacervate microdroplets demonstrated some unique properties,such as selective molecular enrichment,liquid fluidity and dynamic assembly.In this thesis,aiming at the controlled NO generation and release,coacervate microdroplets were developed as the biological carriers for the controlled production of NO through cascade enzymes.Four aspects of research wok have been carried out as follows.1.Construction of erythrocyte membrane-enclosed coacervate microdroplet vesicles and their reduced hemolysis effect.The coacervate microdropts,formed via liquid-liquid phase separation,were enveloped with erythrocyte membrane fragments,which resulted in a significant improvement in the structual stability and hemocompatibility.It is easy for the extraction of erythrocyte membrane because erythrocyte cells have no organelles.Aqueous suspensions of membrane-enclosed coacervate microdropts were produced by the spontaneous interfacial assembly of negatively charged erythrocyte membrane fragments on the surface of preformed positively charged coacervate microdroplets prepared by mixing diethylaminoethyl-dextran chloride and double-stranded deoxyribonucleic acid.Membrane encapsulation led to a change of the surface charge from positive to negative,and an increase of particle size.The structure of membrane-enclosed coacervates was characterized by fluorescence confocal microscopy images.Colorimetry and fluorescence-activated cell sorting demonstrated that membrane encapsulation minimized the direct contact between the coacervate droplets and natural erythrocytes,which in turn significantly improves their stability and hemocompatibility.For example,only 2.6%hemolysis was observed after 90 min at an enclosed coacervate concentration of 0.6 mg·m L^-1.Moreover,the encapsulation of erythrocyte membrane enhances the serum stability of the coacervate droplets,and also extends the blood circulation time,which lays a foundation for further biomedical application in vivo.2.Enzyme-mediated NO production in erythrocyte membrane-enclosed coacervate droplets for vasodilatationCoacervate droplets enclosed with erythrocyte membranes were used as carriers to controllably generate NO for blood vessel vasodilation.In the presence of glucose and hydroxyurea,glucose oxidase inside the coacervate and hemoglobin anchoring on the erythrocyte membrane acted as cascade enzyme for the NO production.The cascade reaction for NO generation involved in the glucose oxidase mediated glucose oxidation,and hemoglobin-mediated peroxidase oxidation of hydroxyurea,in which H₂O₂acted as intermediate.Typically,the enzymatically active membrane enclosed coacervates produced NO levels of up to 1.3μM,which gave rise to a tension force of 5.0 g and a blood vessel relaxation of 38%in vitro aortic vasodilation.The production of NO in vivo was detected to be about 200 n M.The blood pressure and heart rate of rabbits were measured to further verify the expansion effect of dilation on blood vessels.NO is an important physiological regulator that maintains the basic tension of blood vessels.Increasing the concentration of endogenous or exogenous nitric oxide in blood is of great significance for the prevention and treatment of cardiovascular di seases such as thrombosis and hypertension.Therefore,coacervate droplets for delivery of NO were developing into an efficient measure for prevention and treatment of cardiovascular disease.3.In-situ encapsulation of erythrocyte membrane on coacervate microdroplets for oxygen deliveryMixing of erythrocytes and coacervate microdroplets together led to in-situ encapsulation of erythrocyte membrane on coacervate microdroplets.When coacervate microdroplets were incubated with erythrocytes at a certain proportion,the erythrocytes would be fragmentized by hemolysis and then the fragments could be spontaneously adsorbed on the surface of the coacervates for the encapsulation.Compared with conventional methods,which involves the extraction of erythrocyte membrane by a hypotonic swelling method,in-situ encapsulation provides a simple and effective way for the membrane encapsulation.As the hemoglobin present in erythrocytes was a protein responsible for oxygen delivery in organisms,so these membrane enclosed coacervates were qualified the capability for the oxygen delivery.Further experiments showed that erythrocyte membrane encapsulated coacervates could release more oxygen and sustain 9 times longer than that of phospholipid encapsulated coacervates.This work not only provides a simple and fast method for the encapsulation of coacervate droplets,but also provides new ideas for the development of oxygen-carrying materials.4.Spatial distribution of cascade enzymes in coacervates for controlled released of NO and anticoagulant applicationGlucose oxidase and horseradish peroxidase were packaged inside the coacervate,while catalase was immobilized on the periphery of the droplets,which was exploited for the controlled released of NO and anticoagulant application.Dipalmitoyl phosphatidylcholine was used to encapsulate the glucose oxidase/horseradish peroxidase-containing coacervate microdroplets,and catalase was immobilized on the membrane periphery.In the presence of glucose and hydroxyurea,glucose oxidase mediated oxidization of glucose and horseradish peroxidase mediated peroxidation of hydroxyurea initialed the production of NO in the interior of the coacervates.The excess intermediate product of H₂O₂was decomposed by catalase on the phospholipid membrane to H₂O and O₂.Spatial distribution of cascade enzymes allows for the selective release of NO and minimizes the release of H₂O₂.This work provides an important reference for further NO biological applications.