Construction Of Coacervate Protocells And Their Interaction With Living Cells

Cells are the basic unit of morphological structure and functional activities of life realizing a series of life processes such as physiological metabolism,energy conversion and information communications.Protocell,as an artificial cell model,is a vision of the primitive life on earth,and has the characteristics of simple structure and relative stability.It is expected to make up for the shortcomings of the complexity and fragility of real cells and replace dysfu nctional cells with potential applications in cell biology,chemical biology and biomedical applications.And the research of protocell will provide theoretical and experimental basis for the origin of life.However,there are relatively few models of prot ocells at present,and their application in the field of biomedicine has just in the early stage.Based on this,this paper developed a new type of protocell model based on coacervate droplets and carried out research on the interaction with cells,providi ng important references for biomedical applications.The specific research contents are as follows:1.Based on phospholipid surface assembly of coacervate,coacervate vesicles as a new protocell model has been developed.Phospholipid vesicles and coacervate droplets are widely used as protocell models,but their performance and model applicability need to be improved and developed.Based on this,combining phospholipid vesicles and coacervate droplets,a new protocell model has been developed by spontaneo us assembly of the amphoteric phospholipid molecule 1,2-dipalmitoyl-sn-glycero-3-phosphocholine on the surface of preformed polynucleotide/polysaccharide coacervate microdroplets.The surface charge and morphological structure of coacervate were characteri zed by laser scanning confocal microscope,transmission electron microscope and small-angle X-ray scattering,etc.Coacervate vesicles are micron-sized in size,which consist of macromolecules dense internal environment and selective permeability phospholipid bilayer membrane.Photobleaching recovery experiments and fluorescence polarization experiments were used to investigate membrane fluidity.Compared with phospholipid vesicles,coacervate vesicles membrane fluidity decreased.The model has good structural stability and is easy to prepare.It is permeable to molecules below a molecular weight cutoff of ca.4 k Da.Based on this model,a cascade enzymatic reaction system was constructed by encapsulating glucose oxidase(GOx)and horseradish peroxidase(HRP)into coacervate vesicles.The cascade enzyme biometabolism in GCVs mediated by externally added glucose was investigated.The hammerhead ribozyme and the substrate labeled with the fluorescence resonance energy transfer donor-acceptor group were packed into the GCVs.The externally added Mg²⁺mediated ribozyme cleavage reaction was investigated.Quantitative analysis of electrophoresis showed that the ribozyme activity in GCVs increased by about 2.2 times.The results show that coacervate vesicles mediate the efficient biochemical reactions progress by biological enzymes and ribozymes.The coacervate vesicles as a new protocell model,are expected to develop microreactors for biomedical applications.2.Based on the coacervate with enzymatic activity,inva sion and defense behaviors between artificial cells and living cells is established.Establishing the relationship between protocell and living cells is a bridge connecting non-living substances and living substances.Encapsulation of cascade enzymes in coacervate protocell,the invasion and defense relationship between protocell and living cells was established through enzyme-mediated reactive oxygen species(ROS)damage.Coacervate protocell containing GOx was englobed by living cells based on electrostatic interactions.Flow cytometry is used to determine population dynamics in protocell communities.Laser confocal microscopy imaging and cell viability assay investigated the invasion and defense behavior between coacervate and living cells,as well as the enzymatic activation mediated by protocells.The results showed that the coacervate protocells with GOx enzyme activity act as invaders,trigger glucose oxidation and ROS production,and induce the death of living cells.As a defense,the host cell intern alizes catalase(CAT),which mediates the elimination of ROS.The cell viability was restored from 30.0%to 81.0%.This work achieved the regulation of the proliferation ability of living cells through enzymatically active protocells,which provides a new method of regulating cell behavior through protocells.The interaction model between protocells and living cells has been built a bridge model that mediates non-living entities and organism.3.Based on gene transfection mediated by coacerva te protocells,a new method for nitric oxide induction of cell apoptosis has been developed.Coacervate protocells mediate nitric oxide synthase(NOS)plasmid transfection,locally and efficiently produce nitric oxide(NO)leading to cell death,and a new method for NO-induced cell apoptosis has been developed.The NOS plasmid is efficiently loaded in the coacervate protocells,and it is englobed by the host Hep G2cells.The high-efficiency expression of NOS mediated by coacervate protocells promotes the high-efficiency production of intracellular NO,which in turn induces cell apoptosis.The electrophoresis experiment characterizes the efficient load of the coacervate on the plasmid.Confocal imaging and flow cytometry data indicate that plasmids can be effectively released in cells and perform efficient gene expression and translation.Live and dead cell staining experiments and cell viability experiments investigate the effect of NO on cell viability.The results indicate that NO induces cell apoptosis by inducing the overexpression of p53 and Caspase3.This work provides a method for gene transfection mediated by coacervate protocells,and develops a new way to induce apoptosis based on NO.It is proved that coacervate protocells can be developed for cargo delivery systems for biomedical applications.4.Construction of coacervate artificial organelle model for removal of organic dyes.Taking the membraneless organelles from liquid-liquid phase separation in the cell as the bionic object,bio-polyelectrolyte-based coacervate droplet as a membraneless subcellular organelle model is constructed,which contains the reaction route of metabolic enzymes,the removal of Rhodamine B organic dye in the cell is achieved.The GOx and Fe₃O₄ are co-encapsulated inside the coacervate droplets,and the coacervate droplets are electrostatically combined with the host Hep G2 cells and then englobed.Confocal imaging showed that coacervate,as exogenous artificial organelles,can efficiently enrich free Rhodamine B in cells.Glucose,as an endogenous molecule,initiates the Fenton reaction within the coacervate,that is,glucose triggers the GOx and Fe²⁺/Fe³⁺cascade system to generate free radicals,which achieves the effective degradation and removal of Rhodamine B.Cell viability experiments show that artificial organelles based on coacervate can improve cell viability by eliminating organic dyes.This work provides a new artificial organelle model based on coacervate droplets and a new method for the degradation of organic dyes in cells.