Microchip-based Study On The Optimization Of The Preparation Conditions For Uniformized Giant Lipid Vesicles

Since the phospholipid molecules are amphiphilic,spherical vesicles surrounded by one or more lipid bilayers can be formed when the dried lipid film is exposed to the aqueous phase.The formed vesicles are called liposomes.Those vesicles with a size greater than 1 ?m are named giant lipid vesicles.The membrane structure and curvature of giant lipid vesicles are close to those of eukaryotic cells,so this kind of vesicle is an ideal model of cell membrane.It has a wide range of biochemical applications.It can be used to quantitatively study the effects of the component on cell membranes,such as phospholipids and proteins and to study physical properties of cells such as deformation and fusion.Also,it can be applied for gene and drug delivery.However,there are still some problems in the preparation of uniformized giant lipid vesicles,such as residual organic phase and complicated operation,which limits high-throughput related research.This study focused on the development of a simple and easy method for the preparation of uniformized giant lipid vesicles without organic residues.The following research work has been carried out:(1)The method for preparing arrayed lipid film was investigated on the PDMS microarray chip.The lipid solution was directly dropped onto the chip.Due to the difference in height between the lipid film inside and outside of the micro-apertures,the lipid on the surface of the chip can be simply wiped off,and thereby an arrayed lipid film can be obtained rapidly.The effects of the depth and diameter of micro-apertures as well as the concentration of lipid solution were systematically explored.It was found that the micro-aperture depth mainly determines the morphology of the lipid film,and the micro-aperture diameter and the concentration of the lipid solution mainly determine the size of vesicles.By optimizing the parameters,the standard size difference of the prepared giant lipid vesicles was less than 5 ?m.(2)In order to improve the structure of vesicles,the optimized electrical signal parameters and the appropriate concentration of lipid solution for the preparation of giant unilamellar vesicles on ITO glass by electro-formation method were investigated.Three kinds of frequencies(10/500/1kHz),seven amplitudes(1/2/3/5/10/15/20 Vpp)and nine lipid solution concentrations(0.0625/0.125/0.25/0.5/1/2/4/6/8 mg/mL)were systematically explored.It was found that 10 Hz-2 Vpp is the optimized electrical parameter,and 0.25?2 mg/mL is a suitable concentration range for the preparation of giant lipid vesicles.(3)The optimal conditions for the preparation of uniformized giant unilamellar vesicles on the ITO array chip by electro-formation were explored.The effects of the spacing between micro-columns and micro-apertures on the preparation of arrayed lipid film and uniformized giant unilamellar vesicles were systematically investigated.It was found that the spacing between micro-columns was not conducive to the formation of uniformized lipid film and vesicles.The spacing of the micro-apertures can hinder the solution entering the micro-apertures.Moreover,it is not helpful for the formation of uniform vesicles.The optimal structure is the micro-column structure with no spacing between the micro-columns.The standard size difference of the prepared vesicles on a micro-chip with this kind of structure was about 2 ?m.In summary,this study has achieved the preparation of uniformized giant lipid vesicles based on the microarray chip.The main factors such as micro-apertures' geometry(depth,diameter,spacing),the concentration of lipid solution and electric field parameters were experimentally and theoretically discussed.At the end,the corresponding optimal parameters were obtained.All of these can lay a solid foundation for future related research.