| BackgroundGiant Vesicles(GVs)are spherical lipid vesicles with a diameter from1 ?m to 100 ?m.The size and membrane structure of GVs is similar to cells,which makes them widely used for simulation studies of cells and its' inner compartments,structures,and functions of cells and cellular membrane.Electroformation is the most common method applied for GVs preparation.However,the electroformation of GVs is hampered in high ionic solutions(<50 mmol/L),which greatly limits the application of the GVs model in high ionic concentration solutions such as in physiological saline.The suppressing effects of ions on GVs' electroformation are mainly attributed to the inhibition of lipid film and interference of the electric field.At present,the inhibition of ions on the film separation during electroformation of GVs in saline has been effectively improved by adding charged lipids or improving the coating substrate,which promoted the electroformation of GVs in ionic solution successfully.In this study,we propose to improve the conventional electroformation device and optimize the lipid film coating parameters and electric field parameters to establish an efficient method for the preparation of GVs under physiological saline,and to explore the experimental technique and theoretical basis for the study of GVs preparation in high ionic solutions provided by the improved mechanism.ObjectiveThe study is to develop a device and method for effective electroformation of GVs in physiological saline by modifying the electroformation device and optimizing the parameters.Methods1.The modified Teflon frame was produced by adding an insulating septum to the chamber area of the conventional Teflon plate frame,which was then assembled between two Indium Tin Oxide(ITO)electrodes to form a modified electroformation device.The success of the modification was judged by comparing the number and diameter distribution of GVs prepared by electroformation in deionized water and physiological saline using the modified device and the conventional device respectively.2.The relationship between solution ion concentration and GVs formation was investigated by observing the formation of GVs in deionized water and different concentrations of Na Cl solutions using the conventional electroformation device;the impedance of deionized water and different concentrations of Na Cl solutions were then measured separately to determine the relationship between solution ion concentration and impedance;finally,we compared the inter-electrode impedance in solution and the effect of the solution impedance on the inter-electrode electric field intensity,which was theoretically simulated to analyze the possible mechanism by which the modified device promotes the electroformation of GVs electricity in solutions with high ion concentration.3.Dipalmitoyl phosphatidylcholine(DPPC)phospholipids labeled with a lipid bilayer fluorescent probe(3,3'-Dioctadecyloxacarbocyanine perchlorate,Di O)were used at different DPPC concentrations(5 mg/m L,10 mg/m L,20 mg/m L),volumes(5 ?L,10 ?L,20 ?L,40 ?L)and temperatures(25 °C,35 °C,45 °C)were applied to the surface of the ITO electrode to analyze the deposition and homogeneity of the lipids by fluorescence microscopy and to optimize the parameters for the preparation of lipid films on the electrode surface.4.DPPC coatings were prepared in saline using a modified electroformation device with optimized parameters.By comparing the number and diameter distribution of GVs prepared in saline solution under different parameters such as charging times(1h,3h,5h),frequencies(10Hz,100 Hz,1 k Hz,10 k Hz,100 k Hz),electric field intensities(2.5 V/mm,5 V/mm,7.5 V/mm,10 V/mm),the electric field parameters were optimized.Results1.The conventional electroformation device was successfully modified by insulating the chamber of the conventional electroformation device.Under the same electrical parameters and conditions,the conventional device formed a large number of visible spherical vesicles of GVs over 5 ?m in diameter in deionized water,and a similar number and morphology of GVs were seen in the electroformed samples using the modified device.While in normal saline,there was almost no GVs formation visible under the light microscope in the conventional device,but a large number of GVs was observed in the modified device.2.In the conventional device,GVs formation in low concentration(0-0.0009%)Na Cl solutions decreased with the increase of Na Cl concentration,and decreased significantly as Na Cl concentration increased to(0.009%-0.9%).The impedance of Na Cl solutions increased with the decrease of Na Cl concentration.Between the electrodes of the conventional electroforming device,the impedance of deionized water was approximately 10 M? at 40 Hz,significantly higher than that of the normal saline solution which was 200 ?.Between the two electrodes of the modified electroforming device,there was no significant difference between the impedance of deionized water and physiological saline,both exceeding 10 M?.The simulations showed that the electric field intensity between the two electrodes gradually decreased as the impedance of the solution decreased.3.The overall fluorescence intensity of the lipid film gradually increased with increasing DPPC concentration.At lower concentrations(5-10 mg/m L)the fluorescence was more uniformly distributed.At 20mg/m L,a 'wavy' uneven film layer appeared.The overall fluorescence intensity of the lipid film gradually increased with increasing volume from5?L to 40 ?L.At the volume of 5?L to 10 ?L,the fluorescence distribution was uniform under the microscope.At the volume of 20 ?L,the lipid coating began to show a 'crystalline' change in clumps.When the volume was increased to 40 ?L,the 'honeycomb' inhomogeneous film layer appeared.When the electrode was coated at different temperatures,the fluorescence distribution was uniform at 25°C,there was no significant change in the microscopic fluorescence at 35°C,and the lipid coating began to showing more black uncoated areas at 45°C.4.GVs were prepared by electroformation in saline using a modified device at different parameters,and a large number of spherical structured GVs were visible in samples charged at 2.5 V/mm and 10 Hz for 1-3 h;the number of GVs decreased significantly after 5 h.The GVs were formed in all five groups of frequencies(10 Hz,100 Hz,1 k Hz,10 k Hz,100 k Hz)when charged at 2.5 V/mm electric field intensity for 4 h.In the low-frequency range 10Hz-1 k Hz,the number of GVs formed increased with increasing frequency and the morphology was better.When the frequency was greater than 1 k Hz,the number of GVs formed decreased.The number of GVs formed increased with increasing electric field intensity in the range of 2.5-10 V/mm at a frequency of 1 k Hz for 4 h,reaching a maximum of 5 V/mm.Further increasing the electric field intensity,the GVs diameter became significantly smaller,and at an electric field intensity of 10 V/mm,the microscopic GVs morphology was poor and a large number of lipid fragments appeared.ConclusionIn this study,by adding an insulating layer between the two electrodes to modify the electroformation chamber,the decreased electric field intensity was restored,which promoted the electroformation of GVs in physiological saline successfully.Homogeneous lipid films were obtained at a DPPC concentration of 5-10 mg/m L,a coating volume of 10 ?L,and a coating temperature at 35°C.Effective electroformation in physiological saline was achieved in a modified device with electric field parameters of 1k Hz and 5 V/mm within 5 h. |
PDF Full Text Request