Construction Of Artificial Membraneless Organelle With Coacervate Microdroplets For Scavenging Of Reactive Oxygen Species

Organelles are microorgans with specific morphology and function in cytoplasm,which constitute the basic structure of cells and maintain the normal operation of cells.There are two main types of organelles,membranous organelles and membraneless organelles.Among them,membraneless organelles are composed of protein or RNA macromolecules that self-assemble through liquid liquid separation(LLPS)driven by polyvalent interaction.From a chemical point of view,they are Coacervate Microdroplets.The Coacervate Microdroplets have a microcompartment structure,which is a dense environment of biological macromolecules.It has the characteristics of adsorption of small molecules,inclusion of protease and other biological macromolecules,and is the structure and function model of membraneless organelles in cells.The construction of Coacervate Microdroplets in vitro as artificial membraneless organelles and implantation into cells is expected to develop new microorgans with structure and function.They are expected to replace the dysfunctional organelles,which has a potential application prospect in the field of biomedicine.Based on this,in this paper,a membraneless artificial organelle was constructed using coacervate droplets.By coating peroxidase(CAT),the artificial organelle was given the function of scavenging intracellular reactive oxygen species(ROS).Specific research contents are as follows:1.Preparation and structural properties of polyelectrolyte coacervate microdroplet Coacervate microdroplets were prepared by liquid-liquid separation induced by the electrostatic interaction of positive and negative charge polyelectrolyte.Their morphology,liquid fluidity and molecular adsorption effect were characterized.Polydimethyldiallylammonium chloride(PDDA)and polyacrylic acid(PAA)were mixed as positive and negative charge polyelectrolytes in different proportions.The liquid-liquid separation mediated by electrostatic force resulted in the formation of coacervate microdroplets with different surface charges and size morphology.Among them,m(PDDA):m(PAA)=0.8 is the isoelectric point of the coagulated microdroplets,and the size of the microdroplets is the largest(4.9?m).The coacervate microdroplets have good liquid fluidity and present a crowded state of macromolecules inside them,which can efficiently distribute and adsorb small molecular dyes.For example,the distribution coefficients of the coagulant microdroplets for Hoechst,FITC and Cy5 are2506,7886 and 14634,respectively.In addition,the coacervate microdroplet can be used as an excellent reactor.The glucose oxidase mediated glucose metabolism reaction indicated that the coacervate microdroplet enriched the biological enzyme and promoted the improvement of reaction rate and efficiency.This scheme provides a new idea for the design of artificial organelles and the development of intelligent chamber microreactor.2.The application of coacervate microdroplet as membraneless artificial organelle scavenged intracellular reactive oxygenCAT was encapsulated in the coacervate droplets to construct the coacervate loaded with enzyme,which was implanted into SMMC-7721 cells through cell internalization.The coacervate droplets keep liquid form in the cell,still have liquid fluidity,and have the characteristics of mutual fusion.In addition,coacervate microdroplets can effectively remove reactive oxygen species in cells,and the scavenging effect of coacervate microdroplets is related to the amount of internalization in cells(intracellular enzyme concentration).The enzyme concentration in cells is 64 ?g/mL,which can achieve a better scavenging effect of reactive oxygen species.By decreasing the level of ROS in cells,the coacervate droplets can reduce the toxic and side effects of ROS in cells and increase the survival rate of cells to 76.0%.The enzyme-loaded coacervate microdroplet constructed in this work can be internalized by the cell to maintain the microliquid structure and fluidity characteristics,so it is an artificially constructed membraneless organelle structure.This scheme has important guiding significance for the development of artificial organelles and artificial cells.