Protein-based Synthetic Lipid Droplet And Its Dynamic Behaviors

In recent years,various cell-like structures have been constructed through supramolecular self-assembly and a series of mimicries of cell functions and dynamic behaviors have been realized with the rapid development of synthetic biology.By separately integrating various biochemical processes of cells,such as metabolisms of energy,nucleotides,proteins and carbohydrates,the synthetic systems has been further improved to living systems step by step.However,there are still many problems.The structure and function of the synthetic biomimetic cell model are relatively simple and limited.And there are few studies on the coupled dynamic behaviors（such as cell growth,deformation,etc.）of cell metabolism.Thus functional synthetic cells and multi-compartments that integrate various living processes and their interactions are needed to develope the anbiotic substances to autonomic synthetic systems.This thesis intends to design and construct a stable protein-based lipid droplet and synthetic hierarchical cells to realize lipid metabolism and couple the dynamic behaviors,such self-consumption,self-growth,endocytosis,communication and mobility.The main research contents are as follows:First of all,stable protein-based synthetic lipid droplets were constructed to simulate the self-oscillation behavior.The optical interface analysis method was utilised to study the recruit mechanism of the proteins at the oil/water interface,then Pickering emulsion method was used to construct the protein-based lipid droplet.Amphiphilic protein spontaneously recruited at the oil/water interface by reducing the oil/water interface energy,forming a protein-based membrane,effectively separated the oil and water phases then stabilized lipid droplets for more than 4 days.Through research to deepen the understanding of the protein recruitment mechanism on the lipid droplet,it provid a facilition method for constructing protein-based lipid droplets.Based on this method,the triacylglycerols hydrolase lipases were selected as motifs,and the substrate tributyrin and the unreacted PDMS are employed as the mixed oil core to construct lipase-active lipid droplets.Lipase-mediated decomposition of tributyrin at the oil/water interface at room temperature and release the aqueous soluble products glycerol and butyric acid,thus realized the self-consumption behavior of lipid droplets.At 0℃,lipase-mediated consumption of lipid droplet was prevented and substrates spontaneously diffused into the lipid droplets by adding triacylglycerols,thus realized the self-growth behavior of lipase-based lipid droplets.Therefore,the self-consumption and self-growth of the lipase-based lipid droplets can be oscillated by controlling the hydrolysis rate by an appropriate temperature.A stable,active lipid droplet with lipid metabolism function has been constructed,which provides a platform for further research on the complex dynamic behavior of lipid droplets.Next the hydrolysate fatty acid was acted as a signal factor to induce dynamic endocytosis of the membrane in synthetic lipid droplet.Based on Pickering emulsion method,BSA/DMPC lipid droplets were constructed with BSA/DMPC as the basic elements and tributyrin as the oil phase.By introducing lipase,the tributyrin was hydrolyzed in situ on the membrane of synthetic lipid droplets and released butyric acid as a signal factor.The isothermal titration calorimetry was applied to prove the hydrogen bonds between butyric acid and protein.The contact angle was measured to clarify the effect of butyric acid on the interfacial tension between BSA and the water or oil phase,indicating that the cooperation of butyric acid increased the hydrophobicity of the protein.Thereby,stable protein-butyric acid complex-based vesicles were formed,which can be endocytosed into synthetic lipid droplets.Various subtance,such as calcien,lysozyme,BSA,BSA-NH₂,lipase,Au NPs and DNA（～2000 k Da）could be stored in the synthetic lipid droplets through endocytosis,with partition coefficients to 0.84,1.51,2.86,6.31,41.93,62.57 after 10 min and 3.13 after 24h.The responsive release of substances inside the oil core of synthetic lipid droplets was further studied,thermo and near-infrared light responsiveness were employed to release endocytic vesicles.The study provided a feasible mechanism to explanation how proteins exist in the hydrophobic oil core of the lipid droplets,further expanded the dynamic behaviors of synthetic lipid droplets,and opened up new sights for the delivery of synthetic lipid droplets and biomedicine applications.Furthermore,synthetic lipid droplets were introduced into protein vesicles to construct dynamic synthetic hierarchical cells that integrated lipid metabolism,communication behaviors and mobility behaviors.It’s prior to construct proteinosomes loaded with lipase.Sequentially a mixture of tributyrin and PDMS was added into protenosomes with vigorous shaking,thus the oil penetrated the semi-permeable membrane of proteinosomes to construct the lipase-based lipid droplet organells.The lipid metabolism of lipase-based lipid droplets was regulated by temperature to adjust the components of synthetic lipid droplets.The oscillating mobility behavior of synthetic hierarchical cells activated by synthetic lipid droplets due to changed density.Using lipid hydrolysate fatty acid as a signal factor,the communication behavior between binary synthetic lipid droplets was explored by introducing lyticase-based lipid droplets.Then the slightly water soluble triacetin was introduced to form a concentration gradient in aqueous solution,which was used to study the chemotaxis and communication behavior of synthetic hierarchical cells.The research provided a simplified platform for in-depth study of synthetic hierarchical cells.And the design of synthetic hierarchical cell models and the mobility behavior were further expanded.