Fabrication And Functional Study Of Artificial Cell And Cell Colonies Based On Phospholipid

It is still an unresolved question that how the cell life evolved the current complicated biomembrane system and how the biomembrane system ensured cells to function as units or colonies.One of the most important methods to solve this problem was the fabrication of phospholipids based artificial cells or tissue models.It is expected that these models can reproduce the vital procedures of the generation and evolution of cell life to provide clues for the origin and evolution of life,and robust and interference-free platforms for cell biology study.However,compared with the complicated cellular biomembrane system,the present artificial membrane models were too simple to mimic the structure and function of cells and tissues.To solve this problem,we carried out the following studies.Firstly,two methods including modified electroformation and “bicelles to vesicles transformation” methods,were used to form vesicles under physiological conditions.Then,the morphology evolution of bicelles was investigated,and cisternae stacks with organelle-like morphologies were obtained to mimic the function of organelles,which overcome the present challenge to form cisternae stacks-based organelle models.Finally,the assembly of phospholipid assemblies under magnetic field was investigated based on their diamagnetic property,to form artificial tissues with complicated morphologies.Giant unilamellar vesicles(GUVs)electroformation on plasma treated indium tin oxide(ITO)electrode surface was investigated at different amplitudes,frequencies,phospholipid types,NaCl concentrations,and temperature.At fixed amplitude of 2.5 V,GUVs could be formed in NaCl solution at a high frequency range from 100 Hz to 10 kHz.With increased amplitude,this frequency range shifted to higher frequency regions.Zwitterionic,negatively charged and positively charged phospholipids could all form GUVs.With increased NaCl concentrations,the diameter of GUVs firstly increased,and decreased with further increase of NaCl concentration.With the increase of temperature,the diamater of GUVs increased.This method overcome the difficulty to electroform GUVs on ITO electrode in physiological condition,and was applied for the formation of GUVs(unilamellarity>95%)in phosphate buffer,polymerase chain reaction buffer,and cell culture medium.Bicelles with single bilayer and stacked bilayers were formed in alcohol-water mixture during a recrystallization-like cooling process.The influence of alcohol volume percentage,alcohol type,phospholipid concentration,phospholipid type,and cooling rate on bicelles formation was investigated.Phospholipid assemblies with various morphologies were formed from the shape evolution of bicelles.Firstly,during the natural storing of bicelles,the disturbance from ethanol evaporation or temperature change induced pentagon disclinations in the membranes,which drove bicelles transforming into phospholipid cones.For bicelles with smaller bending rigidity and larger diameter,cones with smaller angle tended to form because of the easier introduction of disclinations.Secondly,when single bilayer bicelles were dispersed in water,they transformed into unstable phospholipid bowls.Thirdly,when bicelles were dispersed in water and heated above their phase transition temperature,vesicles with controlled diameter and layer of membranes by the diameter and thickness of bicelles were formed.Finally,stacked bicelles with charged lipids in water transformed into cisternae stacks.The 3D reconstruction from images taken by laser scanning confocal microscope demonstrated that the samples were mixtures of cisternae stacks with different number of helicoids n,including grana-like structure(n=0),rough endoplasmic reticulum-like single helicoid(n=1),and multiple helicoids such as double,triple,and quad helicoids(n?2).With increased percentages of charged lipids and decreased membrane bending rigidity,the average number of n increased.Under the stimulation of solvent condition or oppositely charged nanoparticles,these cisternae stacks could reversibly expand and compress,similar to “breathing” property of some natural cisternae stacks based structures.Based on this “breathing” property,the function of grana in chloroplast was mimicked,and a microreactor was constructed.Phospholipid assemblies were manipulated under non-paramagnetic or paramagnetic environment using magnet.Under non-paramagnetic environment,stacked bicelles and cones with structural anisotropy could align or deform.Under paramagnetic environment,the aggregation of strcturally isotropic GUVs was observed at sites with weak magnetic field intensity around Ni-or Fe-based materials.Based on the latter case,we investigated the assembly on stainless steel mesh in magnetic field.GUVs “colonies” with different morphologies were formed to mimic the structure of tissues.Except for GUVs,the assembly of sephadex,oil droplet,bubbles,and erythrocytes on the stainless steel mesh was also investigated.In this thesis,GUVs as cell membrane models were formed using modified electroformation method and “bicelles to vesicles transformation” method;“breathing” cisternae stacks as organelle models were fabricated by the shape evolution of bicelles;vesicle “colonies” as tissue models were formed by the diamagnetic manipulation of phospholipid assemblies.Novel biomembrane models were provided for the construction of artificial cells and colonies.Theoretical and experimental foundations were established for the assembly from molecule scale to micro-scale.The work hold great potential in the investigation of the origin of life,cell biology,and biomimetics.