Fusion Triggered Enayme-catalyzed Reaction And Protein Expression Inside An Phospholipid-based Artificial Cell

Phospholipid vesicle is a kind of sphere structure self-assembled by amphiphilic phospholipid molecules in aqueous solution,which can be used as a simple cell model due to its similar structure and components to real cells.Phospholipid vesicles can be used to mimic cellular metabolic processes,including enzyme-catalyzed reaction,protein expression and so on.The fusion of phospholipid vesicles enables mass transmembrane transports,which facilitates the signal communication among cells.In this thesis,enzyme-catalyzed reactions and protein expression were achieved inside phospholipid vesicles based on fusion between giant unilamellar vesicles(GUVs)and large unilamellar vesicles(LUVs).GUVs doped with 10 mass% positively charged phospholipid were prepared by emulsion method with a surface zeta potential of 25.43 ± 0.87 m V.LUVs doped with 5 mass%?10 mass%?15 mass%?20 mass% negatively charged phospholipid,respectively,were fabricated by extrusion method with corresponding surface zeta potentials of-5.46 ± 1.24 m V?-7.11 ± 0.20 m V?-9.95 ± 0.94 m V?-19.10 ± 1.31 m V.After mixing by volume ratio of 1:1 of the two different phospholipid vesicles,fusion between GUVs and LUVs through electrostatic interaction was achieved.The results showed that fluorescence intensity of GUVs increased with the increasing proportion of negative phospholipid in LUVs,meanwhile the balance time of fusion decreased with the increasing proportion of negative phospholipid in LUVs.Excess amount of negatively charged phospholipid in LUVs would result in the instability and rupture of GUVs during fusion process.Thus,the optimal ratio was determined to be 10 mass% positively charged phospholipid in GUVs and 10 mass% negatively charged phospholipid in LUVs.In the thesis,two enzyme-catalyzed reactions based on fusion of GUVs and LUVs were demonstrated.Glucose Oxidase(GOx)?horseradish peroxidase(HRP)and Amplex Red were encapsulated in GUVs by emulsion method,while glucose molecules were encapsulated in LUVs by extrusion method.After the fusion of GUVs and LUVs,GOx catalyzed glucose to generate hydrogen peroxide,which subsequently oxidized the Amplex Red to generate the resorufin with red fluorescence by the assistant of HRP.The other enzyme-catalyzed reaction is as follows.Nicotinamide adenosine dinucleotide(NAD~+)and the glucose-6-phosphate dehydrogenase(G6PDH)were encapsulated in GUVs by emulsion method,while glucose-6-phosphate(G6P)was encapsulated in LUVs by extrusion method.After the fusion of GUVs and LUVs,the G6 P was catalyzed by G6 PDH to enable NAD~+ becoming nicotinamide-adenine dinucleotide(NADH)with blue fluorescence.The vesicle fusion experiment was further applied for the GFP expression by E.coli inside GUVs.The E.coli were encapsulated in GUVs by emulsion method and Isopropyl ?-D-thiogalactoside(IPTG)was encapsulated in LUVs by extrusion method.After fusion of GUVs and LUVs,IPTG could induce GFP expression by E.coli inside GUVs.