Study On Encapsulation Of Yeast Cell With Functionalized Shells Based On Biomimetic Mineralization And Visible Light-induced Surface Graft Polymerization

Cell surface is the critical part of the cell to interact with external environmental substances and generate various complex functions.The introduction of artificial shell layer on the surface of single yeast cells can not only protect cells against adverse environmental interference,but also endow them with a completely new surface structure and function,which has promising applications in biosensors,drug delivery,cell therapy,regenerative medicine,bio-catalysis and tissue engineering.The development of low cytotoxicity modification strategies is a hot direction for single cell coating research and is of great significance for the development of practical living cell-related technologies.However,most of the currently developed artificial shells are grown directly from the cell surface,and the direct contact between the shell layer and the cell surface is not conducive to the maintenance of cellular activity.Based on this background,this thesis developed a method to prepare yolk-shell structure encapsulated yeast cells by using biomimetic mineralization and visible light-induced surface graft polymerization,introducing a void layer between the polymer shell and yeast cells.The main research contents and results of the paper are as follows.1.Preparation of calcium carbonate shell on the surface of yeast cells using a biomimetic mineralization method.Firstly,PDADMAC/PAA four-layer polyelectrolyte layer was prepared on the negatively charged yeast cell surface by a layer-by-layer self-assembly method through alternating electrostatic adsorption.On this basis,the deposition of calcium carbonate shell on the yeast cell surface was achieved by the enhanced biomineralization induced by polyelectrolyte layers.The effects of types of regulating additive,PAA concentration,calcium chloride and sodium carbonate concentrations,calcium ion adsorption times and deposition times on the morphology,thickness and integrity of the calcium carbonate layer were investigated.When1.5×10^-4M of PAA was used as a regulating additive,30 m M of calcium chloride to sodium carbonate concentration ratio,1.5 h calcium ion adsorption time and 2 h deposition reaction were used,complete and homogeneous deposition layer（yeast cell@Ca CO₃）could be successfully prepared on the surface of yeast cells.2.A new strategy was developed for the preparation of encapsulated yeast cells with yolk-shell structure.Firstly,the yeast cell@Ca CO₃@poly（PEGDA）with cross-linked polymer shell was prepared by visible light-induced graft cross-linking polymerization of PEGDA on the surface of yeast cell@Ca CO₃adsorbed with PEI.Further,encapsulated yeast cells with hollow polymer shell layer with yolk-shell structure（yeast cell@hollow poly（PEGDA））were prepared by removing the calcium carbonate mineralization layer.The effects of different light irradiation times and light intensities on the thickness and integrity of the poly（PEGDA）coating layer were investigated.The results showed that the complete poly（PEGDA）layer could be obtained at 15 m W/cm²light intensity and 20min light irradiation time.SEM showed that the surface of yeast cell@Ca CO₃consisted of nanoparticle accumulation with a rough surface;after grafting poly（PEGDA）,the cell surface became homogeneous,complete and relatively smooth;the surface was folded and the roughness was relatively increased due to the loss of the support of inorganic layer after removing calcium carbonate layer.The thickness of the graft layer was 200 nm observed by TEM.The chemical structure of the hollow poly（PEGDA）shell layer was confirmed by FTIR and XPS.The results of flow cytometry analysis showed that the cellular activities of yeast cell@Ca CO₃@poly（PEGDA）and yeast cell@hollow poly（PEGDA）were 95.43%and 98.81%,respectively,indicating that the method is very suitable for in situ reactions in the presence of live cells.The results of yeast cell proliferation curves showed that the lag phase of yeast cell@hollow poly（PEGDA）was delayed to about 20-28 h after 20 min-40 min grafting reaction under 10 m W/cm²-15 m W/cm²light intensity,indicating that the introduction of polymeric coating layer could effectively inhibit the proliferation of yeast cells.After 30 days of low temperature storage,the cellular activity of yeast cell@hollow poly（PEGDA）was 79.82%,while the native yeast cell activity was significantly reduced to only 21.26%,indicating that the introduction of the hollow poly（PEGDA）shell layer significantly enhanced the storage stability of yeast cells.3.Using the above method,yeast cells with a functional cross-linked PAAS hollow shell（yeast cell@hollow PAAS）were prepared using AAS as the monomer and PEGDA as the crosslinker.The yeast cell@hollow PAAS could be prepared when the concentration of AAS was 0.2 g/m L,the crosslinker concentration was 5 wt%of AAS,the light intensity was 15m W/cm²,and the light irradiation time was 20 min.The successful coating of the hollow PAAS graft layer on the yeast cell surface was also confirmed by SEM and FTIR characterization.The viability of yeast cell@hollow PAAS was 96.39%by flow cytometry,which again confirmed the superiority of this method in maintenance of cell viability.The ability of the coated cells to resist lyticase was demonstrated by characterization of proliferation curve test,and the introduction of polymeric coating layer can effectively inhibit the proliferation of yeast cells.Encapsulated yeast cells with surface shell layer coupled with urease were prepared by the condensation reaction between the carboxyl groups on the surface of yeast cell@hollow PAAS and urease.The loading capacity of urease could reach 2.18×10^-5μg per yeast cell.