@misc{oai:ir.soken.ac.jp:00000238, author = {金, 勇勲 and キム, ヤンフン and KIM, Yong Hoon}, month = {2016-02-17}, note = {The combination of silicon technology with the cell biological functions is to be an attractive research field for the development of scientific and technological applications including the in vitro study of the fundamental properties of biological membranes and medical diagnosis and the screening for the new drug discovery. Supported membranes are generally prepared by two methods; Langmuir-Blodgett (LB) method and vesicle fusion method. The former is useful for the formation of the hybrid type lipid bilayer comprised of a different type of lipid monolayers. The latter, more convenient and prevail, has several advantages with respect to Langmuir-Blodgett method in a viewpoint of easy deposition, high area selectivity, and defect free high quality membrane formation in a limited area. In this method, Ca²⁺, as a facilitating material, is used to form the lipid bilayer by the vesicle fusion. However, addition of Ca²⁺ gives rise to the limitation to utilize supported membranes for the application areas in relation to cell physiological phenomena. In this thesis, he focused on the addition effects of cholesterol in LB method and the Ca²⁺ free vesicle fusion method for the lipid bilayer formation.
　In LB method, the addition effects of cholesterol on the dipalmitoylphosphatidylcholine (DPPC) monolayer have been investigated by AFM and IRRAS, since Cholesterol, one of the major components in cell membranes, is a regulator to maintain biological and physical properties of the membranes including permeability, fluidity and mechanical strength as well as enhances the resistivity of a supported membrane on a silicon substrate.
　In the analysis of pressure (π) -area (A) isotherms for monolayers with different molar cholesterol concentrations (C_chol) (10, 20, 30, and 35%) at the air-water interface, with addition of cholesterol, the shift of the isotherms toward the left side (small area per molecule region) and the disappearance of plateau region indicate the condensing effects governed by heterogeneous hydrophobic interactions between DPPC and cholesterol.
　For AFM images of the DPPC/cholesterol monolayers varied with C_chol(0~35%) transferred onto mica surfaces at the 10 mN/m deposition pressure, there are two stages in the transformation from pure DPPC (liquid crystalline (LC) + liquid expanded (LE)) to DPPC/cholesterol mixture (liquid ordered (LO)) in the C_chol range of 0~35％: at C_chol from 0% to 10%, ,,_LC only slight decreases in spite of the drastic change in the morphology and at C_chol between 10% and 35%, ,,_LC decreases accompanied with the appearance of the depletion areas, which are cholesterol rich domains. At the initial stage of the cholesterol addition (5%), cholesterol molecules probably distribute at the boundary of the LC domains. After the boundary region is saturated with cholesterol, excess cholesterol gathers at the interface with LE, which is observed as the depletion area or diffuses into the inside of the LC domain.
　In the IRRAS spectra of the DPPC, DPPC/cholesterol mixtures (10, 20 and 35%) and cholesterol monolayers prepared at the 10 mN/m surface pressure, addition of 10 mol % of cholesterol caused the shift of the CH₂ vibrational modes from 2917 to 2922 cm^-1 for ,,as(cH₂) and from 2848 to 2852 cm^-1 for ,,s (CH₂), and also caused the appearance of ,,as(CH₃) at 2964 cm^-1. The blue-shifts of ,,as(CH₂) and ,,s(CH₂) indicate the disordering of the conformation of the DPPC acyl chains due to the weakened lateral hydrophobic interaction and the reduction of the trans population, whereas the position and the shape of the newly appeared ,,as(CH₃) indicates that terminal methyl groups are ordered similarly to that in the solid-like gel phase. The shoulder of ,,as(CH₂) at 2937 cm^-1 is assigned to the Fermi-resonance between ,,s (CH₃) and the overtone of the asymmetric deformation ,,as(CH₃). Addition of cholesterol also caused the broadening of the ,,as(CH₂) and ,,s(CH₂) peaks. The broadening at C_chol= 10% will be due to the heterogeneous distribution of cholesterol in the LC domains between the edge and the center. At the higher cholesterol concentration (C_chol ＞25%), at which the mobility of DPPC molecule increases, the fluidity is a major contribution to the peak broadening. On the basis of above results, I suggest that the transformation from the co-existing LC and LE phases on the pure DPPC monolayer to the homogenous LO phase on DPPC/cholesterol proceeds through two stages: initial drastic changes in the surface morphology and the conformation of the DPPC acyl chains below 10% cholesterol, and the gradual homogenization of the morphology towards the liquid ordered phase up to 35% cholesterol.
　In vesicle fusion method, the effect of the electrostatic attractive force between vesicles and the substrate surface on Ca²⁺ free supported lipid bilayer formation has been investigated by using atomic force microscopy and fluorescence microscopy.
　In a typical of fluorescence images of giant vesicles (GUVs), GUVs containing neutral lipids were aggregated each other, while GUVs including negative-charged lipids were individually segregated. The aggregation is due to the non-charge inter-vesicle attractive interaction. The segregation is from the inter-vesicle charge repulsion induced by the negative-charged lipids, which interrupts the vesicle -vesicle aggregation. The positive-charged surface was prepared by the monolayer deposition of 3-aminoprophyldimethylethoxysilane (APS) on the SiO₂ surface. The observed value of water contact angle (WCA) for the bare SiO₂ surface was ~10⁰ and it changed to ~51⁰ after the surface modification by APS.
　When the negative-charged GUVs were incubated without Cu²⁺, extremely low surface coverage of lipid bilayer was observed on the bare SiO₂ surface. In the presence of Ca²⁺, the high surface coverage of lipid bilayer was observed when Ca²⁺ was added before incubation. The remarkable difference in the coverage of the lipid bilayer on the SiO₂ surface is explained in terms of the adsorption of GUVs. As to the formation of the lipid bilayer by vesicle fusion method, the adsorption is an initial step. In the absence of Ca²⁺, the electrostatic repulsion between the surface and the vesicles was induced during incubation, which results in very low surface coverage of the lipid bilayer. In case of the positive-charged surface modified by the APS monolayer deposition, the high surface coverage of the lipid bilayer was obtained through the electrostatic attractive force between vesicles and the surface. The strong electrostatic attractive force between vesicles and the surface enhances the stable adsorption of the negative-charged GUVs, which promotes the lipid bilayer formation. The rupture of GUVs is induced by the interaction between GUVs and the SiO² surface, almost without influence of the supported GUV-GUV interactions. And, in AFM observation, the thickness of the water layer between SLB and the surface decreases by the surface APS modifications., 総研大甲第889号}, title = {Formation of supported membranes and their characterization by atomic force microscopy, fluorescence microscopy and IRRAS}, year = {} }