Coacervates Formation Based On Vesicle Fusion

Coacervates are membraneless organelles in cells,which can be used to mimic primitive cells.Membrane fusion is a ubiquitous phenomenon in the cell,which plays important biological functions.The membrane fusion of phospholipid vesicles can be achieved through fusion peptide,electrical stimilus and electrostatic interaction.In this thesis,vesicles with different electrical properties were used to induce membrane fusion through electrostatic interaction.After fusion,their contents were mixed to generate coacervates inside the fused vesicles.By adjusting the concentration and ratio of poly(diallyl dimethyl ammonium chloride)(PDDA)and adenosine triphosphate(ATP),coacervates with different size were prepared.When the coacervates were composed of ATP:PDDA=1:4(PDDA=80 m M),large coacervates were formed,which was easy to observe as a compartment for chemical reactions.When the coacervates were composed of ATP:PDDA=1:16 or 1:2(PDDA=80 m M),small coacervates were formed,which was suitable for encapsulating inside vesicles.The coacervates can interact with the positively charged vesicles,causing their aggregation inside the coacervates.Furthermore,the cascade enzyme reaction was realized inside the coacervate as follows: hydrogen peroxide was generated with the assistant of glucose oxidase,which then oxidized Amplex Red to form resorufin with red fluorescence catalyzed by horseradish peroxidase.Giant unilamellar vesicles(GUVs)were prepared using 1,2-dilauroyl-sn-glycerol-3-choline phosphate(DLPC)/dioleoylphosphatidylserine(DOPS).Small unilaminar vesicles(SUVs)were fabricated using 1,2-dioleoylphosphatidylcholine(DOPC)/(2,3-dioleoyl-propyl)-trimethylamine(DOTAP)(1:X mg/mg).DOTAP is a positively charged phospholipid.The doping mass ratio of DOTAP(DOTAP:DOPC)was 0.1:1,0.15:1,0.35:1,1:1,respectively.Under the above conditions,SUVs and GUVs were fused together.It was expected to encapsulate ATP in GUV and PDDA in SUV,and generated coacervates after the fusion between GUVs and SUVs.This experiment has not been carried out due to the COVID-19 pandemic.