The Construction Of Enzyme Loaded Artificial Cells And Their Effects On Hepatocytes

The integral structure and function of cells is still not fully understood.Even the simplest living cells in nature are extremely complicated in structure and function,while the emergence of artificial cells has guided new directions for the exploration of cell structure and function.In the past few decades,the research of artificial cells has made remarkable achievements in the fields of cell biology,molecular biology,supramolecular chemistry and biomedicine.Due to the similarity of the phospholipid assembly to the structural components of the cell membrane,the multiple advantages of the mechanical stability,chemical versatility,and biodegradability of the polymer carriers,studies on phospholipid assemblies and polymer carriers as artificial cells become a hot topic in bionics.However,the current construction methods of artificial cells are immature in many aspect,compared with the natural biological cells,the present artificial cells are too simple to simulate complex cellular structures and functions.Moreover,there is no report on the interaction between organelles in artificial cells at present,and the application of artificial cells in more complex physiological environments and the interaction of artificial cells with biological cells also need to be explored.Therefore,targetting these issues,the artificial cells are constructed in several ways to conduct systematic study in this thesis.1,2-dioleoyl-snglycero-3-phosphocholine（DOPC）as one of the cell membrane components in nature is selected to build artificial cells.The lipid microtubes and giant unilamellar vesicles（GUVs）are formed simultaneously using a nonhomogeneous electric field generated by point-to-plane electrodes.The underlying reason of this phenomenon is analyzed using COMSOL software.The pulling force（F）to drag GUVs into lipid tubes induced by fluid flow,and the critical force（Fc）to prevent GUVs from deforming into lipid tubes induced by electric fields play main roles in the formation of lipid assemblies.The GUVs are obtained if F is less than Fc,whereas the lipid microtubes are formed if F is larger than Fc.Additionally,the relationship between the electrode distances and the induced GUVs and lipid tubes ratios（GT ratio）is investigated using simulation and confirmed by experiments.The results show that the GT ratio increases as the distance between electrodes became larger.Surface attached GUV microarrays are produced by the combination of microcontact stripping technique and electroformation method using a point-to-plane electrode system and polydimethylsiloxane（PDMS）stamps.The size of the GUV is similar to the feature size of PDMS stamps that utilize in the stripping step.Therefore,The size of the GUV microarray are controlled by the size of the PDMS stamp.The GUVs are obtained with the diameter of 27.87±2.22μm,64.06±3.08μm and 82.25±5.76μm by using 20μm,50μm and 85μm stamp,respectively.GUV arrays also can be created using a variety of lipids from single lipid to ternary lipid mixtures.DOPC GUV arrays are chosen as artificial cells for further studies because they present the best quality.The GUV array also serve as a good platform to encapsulate biomolecules（DNA etc.）,simulate transmembrane transport of substances as well as simulate cellular metabolism.The release of carboxylfluorescein（CF）from GUVs demonstrate a melittin concentration dependent manner.The diffusion coefficient（D）are 0.37×10^-11,0.36×10^-11,0.54×10^-11,1.10×10^-11,1.74×10^-11,2.31×10^-11 and 3.62×10^-11 m²/s for 0,0.5,1.0,2.0,3.0,4.0 and 5.0μM melittin,respectively.Horseradish peroxidase（HRP）containing GUV arrays were used to mimic cellular metabolism by exposing them to the substrates of small molecules of H₂O₂ and o-PD to yield fluorescent2,3-diaminophenazine（2,3-DAP）.Inspired by the compartmentalized structure of eukaryotic cells,an alginate-based complex multi-compartment artificial cell with both artificial organelle(catalase loaded liposome,L_Cat)and real nuclei（pNuc）from RAW 264.7cells was built.pNuc are able to synthesize mRNA,while L_Cat is capable of converting hydrogen peroxide（H₂O₂）to oxygen and water.Compared with the artificial cells without L_Cat or in the absence of H₂O₂,artificial cells containing both natural pNuc and synthetic L_Cat show significantly higher mRNA production in the presence of H₂O₂.It indicates that oxygen produced by local enzymatic reaction is able to promote the synthesis of mRNA.The design of this novel artificial cell containing natural and synthetic organelles provides an opportunity as hypoxic models or for cell-free protein synthesis.The particle（P1）arrays are prepared by microcontact printing technique（μCP）,and then the adhesion and proliferation behavior of human umbilical vein endothelial cells（HUVEC）and hepatocytes（HepG2 cells）on the patterned substrates are evaluated.The adhesion and proliferation of HUVEC on the P1 arrays depend on the stability of P1 attachment to the substrates.The HUVEC displace P1when P1 is weakly attached to the substrates.However if P1 is strongly attached to the substrates,particle-free areas are preferred for cell adhesion.The HepG2 cells adhered and proliferated on P1 arrays without preference for specific locations or destruction of the particle arrays within 48 h.Using a layer-by-layer assembly technique,glucose oxidase-loaded liposomes(L_GOx)are immobilized on the surface of nanoparticles by polymer coatings to construct a complex multi-compartment artificial cell.The artificial cell arrays are subsequently prepared as the same method as before.The activities of artificial cell in solution and on patterns are depending on the number of liposome assembled.The artificial cell arrays are co-cultured with HepG2 cells.HepG2 cells adhering to patterned artificial cell surfaces in the presence of glucose exhibited more dead cells due to the local production of H₂O₂ compared to cells attached to enzyme-free carriers arrays.It provides important guidance for the design and construction of complex multi-compartment artificial cells,and has potential in the field of cell therapy.