Construction And Functionalization Of Coacervate/proteinosomes And The Capturing Of Bacteria

Mimicking the structure and function of the native cells has been the focus over the years for researchers in the molecular life sciences.Along with the rapid development of synthetic biology,the spontaneous self-assembly and integration of molecular and functional building blocks into cell-like assembly with cellular structure and function has gained tremendous interest.These cell-like assembly models have been widely used in biotechnology,biomedicine and environmental science,and provided important theoretical basis for understanding cellular mechanisms and the origin of life.To date,a majority of researches mainly focused on the functional design of individual cell-like assembly,while the research models were relatively rare that investigated the interactions between the assembling entities and environment,especially the regulable interactions between the assemblies and native living cells.To make an in-depth simulation towards the cellular interactions and further explore the relevant mechanisms,the designed and constructed functional coacervate microdroplets/proteinosomes based on the spontaneous self-assembly of different building blocks was fabricated to investigate their various interactions with bacteria,which provided the new idea to the development of the interactions within cell-like assembly and native cells hybrid microsystems,and the design of biomimetic materials with advanced cell-like functions and applications such as in sterilization.The main research contents are as follows:The positively charged and thermosensitive coacervate microdroplets were prepared by utilizing zwitterionic polymer（PST）with upper critical solution temperature（UCST）behavior and positively charged quaternary ammonium salt（PMEDAB）as building blocks that underwent phase separation.The fact that the electrostatic and hydrophobic interactions as the main driving force for phase separation was proposed and verified.Dynamic regulation of the formation/dissociation of coacervate could be realized by adjusting temperature.At room temperature,the PST molecular chain was curled,not only was the stability of the coacervate microdroplets improved because of physical crosslinking in the interior,but also the cytoplasm-like properties of the coacervate including the sequestration capacity and the mobility were elegantly maintained.Furthermore,via the electrostatic interactions between the positively charged coacervate microdroplets and bacteria realized the capture of the coacervate towards bacteria,and the capture efficiency could reach 99.9%（the ratio of bacteria to coacervate microdroplets was less than 140:1）.Besides,the degree of aggregation between the coacervate microdroplets and bacteria could be regulated by adjusting quantity ratio of the hybrid microsystems.In addition to macroscopically studying the capture and aggregation of the coacervate microdroplets towards bacteria by using the electrostatic interactions between coacervate microdroplets and bacteria.Based on this,a new method that the coacervate microdroplets were reconstituted with natural cellular wall fragments was developed to mimic the behavior of macrophages,and the more complex interaction between the coacervate microdroplets and bacteria was established,which demonstrated the selective capturing and programmed phagocytosis of reconstituted coacervate microdroplets towards bacteria in microscopically.The membrane-bound compartmentalizing coacervate microdroplets was constructed with the spontaneous assembling of the natural yeast cellular wall fragments around the polysaccharide based coacervate microdroplets.The fact that the high interfacial adsorption energy of the employed yeast cellular wall fragments and the electrostatic interactions as the main driving force for the spontaneous assembly at the interface of water and coacervate was proposed and verified.The inherent properties of the reconstituted coacervate microdroplets including the selective sequestration capacity and the molecule crowded environment were elegantly maintained,and the stability was well improved.Moreover,the biological property of the yeast cellular wall was well maintained in the reconstituted coacervates,allowing the preferential capturing of the coacervates microdroplets towards the Fim H-expressing bacteria based on specific interaction of mannans on the yeast cellular wall fragments with Fim H,the capture efficiency was about 46%.By regulating wetting properties of the interior coacervate toward the captured E.coli and the shear force,the compartmentalized coacervate microdroplets could engulf about 36%of the captured E.coli and kill them.On the basis of the capture of bacteria of the two types of coacevate assembly models,in order to further provide and enrich interaction models,in this part,the cell-like assembly based on the proteinosomes was constructed to regulate the complex interaction between proteinosomes and bacteria,where the reversible capture process of proteinosomes towards bacteria was demonstrated.Moreover,the captured bacteria can induce proteinosomes to release fungicides to kill itself.The positively charged and thermosensitive proteinosomes with loaded acid-sensitive hydrogel was constructed by utilizing the coupling of mercaptothiazoline-activated PNIPAAm-co-PMEDAB polymer chains with primary amine groups of cationized BSA-NH₂.The hydrogel was composed of aldehyde-functionalized dextran（Dex-CHO）,BSA-NH₂ and bactericidal L-arginine grafted chitosan oligosaccharides（COS-Arg）cross-linked by the formation of Schiff base bonds.The experiment showed that,at 37℃ and in deionized water solution,the negatively charged living organism E.coli could be highly adsorbed onto the surface of the constructed hydrophobic and positively charged proteinosomes.Quaternary ammonium salts on the surface of proteinosomes could destroy the cell membranes of bacteria that adhered,and cause the bacterial death.With the amount of attached E.coli increasing,the E.coli colonization on the surface of the proteinosomes would reduce the p H of local environment through the metabolism of the E.coli,leading to the rupture of Schiff base bonds in hydrogel,and then the loaded COS-Arg inside the proteinosomes would be released to kill E.coli.Subsequently,more than 81%of the captured E.coli could be desorbed from the surface of the proteinosome by cooling down the temperature to 25℃ with the addition of phosphate buffered saline（50 m M,p H 7.4）,due to the changes of the hydrophobicity and the electrostatic attraction of the proteinosomes.Besides,by controlling the temperature and ionic strength,the programmed capture and release process of proteinosomes towards bacteria was realized,and this process could be recycled by three times.