Composite membranes of polymeric and fluid lipid bilayers K. - PowerPoint PPT Presentation

Composite membranes of polymeric and fluid lipid bilayers K. Morigaki, 1,2 T. Okazaki, 2 Y. Tatsu, 2 and H. Imaishi 1 1 Research Center for Environmental Genomics, Kobe University 2 National Institute of Advanced Industrial Science and Technology (AIST) 1

Membrane functions & biomedical applications Biomedical Membrane functions applications • Signal transduction • Drug development • Energy conversion • Diagnostics • Immune system • Biosensors • Cell-cell recognition Measuring and manipulating the functions of cellular membranes at the molecular level Synthetic model membranes 2

Roles of the model membrane systems (biological membrane) (model membrane) ca. 1900 lipidic Langmuir monolayer 1925 bilayer structure lateral mobility 1960s lipid vesicles (liposomes) asymmetry planar bilayer (BLM) permeability integral/ peripheral 1972 membrane proteins (fluid mosaic model) planar bilayer (substrate supported) more complex architecture From giant vesicles (microdomains, caveolae, 1980s membrane skeleton, etc.) New model More complex structures membranes and functions 3

Model systems of the biological membrane substrate supported planar lipid bilayer (black lipid vesicles lipid bilayer lipid membrane: BLM) (liposomes) 1. Mechanical stabilization 2. Surface specific analytical techniques 3. Integration by micro-fabrication techniques 4

Generation of complex model membranes Patterned model membrane Supported bilayer polymeric bilayer natural lipid bilayer Micro-fabrication & Self-assembly 5

Fabrication of patterned lipid bilayers Polymerizable lipid (1) Monomer lipid H 2 O O bilayer (LB/ LS method) O O H O O P O + N O O Polymerization h ν (2) Photopolymerization R1 R1 R1 h ν (254 nm) R1 with a mask R2 R2 R2 R2 n (3) Removal of monomer lipids (4) Incorporation of new lipid bilayers 6

Polymerization of lipid bilayers Synthetic polymerizable lipids: Originally developed for the stabilization of liposomes for drug delivery applications (Ringsdorf, O’Brien, Chapman, Regen, Tsuchida..) h ν Δ Diacetylene lipid (DiynePC) R1 R1 R R O O O O O H O O O O P O + N O O R1 R1 R1 R2 h ν (254 nm) R1 R2 R2 R2 R2 R2 n 7

Polymerization in lipid bilayers UV/visible absorption spectra Diacetylene lipid (DiynePC) 0.010 0.008 O Absorption (AU) polymer 0.006 O O H O O P O + 0.004 N O O 0.002 0.000 monomer h ν R 1 R 1 R 1 R 1 R 1 -0.002 R 1 R 1 R 1 R 1 R 1 200 300 400 500 600 Wavelength (nm) Fluorescence spectra R 2 R 2 R 2 R 2 R 2 R 2 R 2 R 2 R 2 R 2 Unique features: 1. Topochemical polymerization 2. Conjugated polymer backbone 8

Patterned polymerization of DiynePC bilayers frequency 0 2 4 6 height [nm] A polymerized Diyne-PC bilayer AFM observation of a lithgraphically after lithographic UV light exposure polymerized DiynePC bilayer. The polymeric and removal of monomers. The bilayer consist small domains. The size of scale bar corresponds to 50 μ m. corrals is 20 μ m. Okazaki et al. Langmuir 25 , 345 (2009) 9

Fluid bilayers in polymeric bilayer corrals A B E - + C D Fluid lipid bilayers (egg-PC/ TR-PE) can be incorporated into the corrals, and application of an electric field induces concentration gradients. The size of corrals is 50 μ m. 10

Roles of polymeric bilayer matrices Unique feature Polymeric and fluid bilayers are forming a continuous hybrid membrane Roles of polymeric bilayers • Controlling the lateral distribution of molecules • Stabilization of the model membrane 11

Confinement of fluid bilayers in the corrals A B C D 4 min after the photobleach The bleached corral became homogeneously dark, indicating that lipid molecules are diffusing laterally within the corral. The scale bar corresponds to 50 μ m. 12

Controlling the composition of bilayers Large UV dose Small UV dose Polymeric and fluid bilayers can be integrated as sub-micrometer domains by modulating the degree of polymerization. JP-Patent 4,150,793 (2008) Morigaki et al. Langmuir 20 , 7729 (2004) 13

Size distribution of polymeric bilayer domains 200 150 frequency 100 50 0 0 5 10 15 20 25 30 radius of polymeric domains (nm) 1 x 1 μ m The size distribution of the polymeric bilayer domains observed after the SDS treatment. The radius of domains was plotted in a histogram, assuming a circular shape. The influence of finite AFM probe curvatures is not corrected. Okazaki et al. Langmuir 25 , 345 (2009) 14

Polymeric bilayers with different UV doses 2.5 J/cm 2 1.5 J/cm 2 4.0 J/cm 2 AFM images (1 x 1 μ m 2 ) of polymeric bilayers prepared with different UV doses. The samples were observed after the removal of monomers by SDS treatment. Okazaki et al. Langmuir 25 , 345 (2009) 15

The amount of polymerized bilayers 5 1.0 Polymerized lipid (nm) 4 Area fraction from AFM 0.8 3 0.6 2 0.4 1 0.2 0.0 0 0.0 0.2 0.4 0.6 0.8 1.0 0 1 2 3 4 5 6 7 Area fraction from ellipsometry 2 ) UV irradiation dose (J/cm Film thickness after the 0.1M SDS The area fractions of polymeric bilayers treatment (ellipsometry) from AFM images were plotted versus those from the ellipsometry and fluorescence microscopy. The amount of polymerized bilayers can be controlled by changing the applied UV dose for polymerization Okazaki et al. Langmuir 25 , 345 (2009) 16

Composition of polymerized and fluid bilayers Fluorescence intensity (normalized) 1.0 (A) Polymer (B) Fluid 0.8 0.6 0.4 2 5 2 5 0.2 0 3 0 3 0.0 0 1 2 3 4 5 Polymerized lipid (nm) Fluorescence microscopy images of a patterned hybrid SPB with spatially varied degree of polymerization ( UV doses shown in The amount of fluid bilayers J/cm 2 ). (normalized to full coverage) and (A) Fluorescence from polymeric DiynePC. (B) polymeric bilayers (Determined by Fluorescence from fluid bilayers. The scale fluorescence microscopy) bars correspond to 100 μ m. The amount of fluid bilayers incorporated changed linearly with the amount of polymeric bilayers, suggesting the formation of a composite membrane. Okazaki et al. Langmuir 25 , 345 (2009) 17

Obstructed diffusion by polymeric membranes (a) 1.5 J/cm 2 * ) Relative diffusion coefficient ( D 1.0 Fitting to the (b) 2.5 J/cm 2 free-area model a 0.5 b (c) 4.0 J/cm 2 c 0.0 0.0 0.2 0.4 0.6 0.8 1.0 Area fraction of obstacle ( c ) Diffusion coefficient of TR-PE was plotted as a function of the amount of polymeric bilayer in the composite membrane. Diffusion coefficients were determined by the fluorescence recovery after photobleaching (FRAP) method. Okazaki et al. Langmuir 25 , 345 (2009) 18

Phase separation of lipid membranes Sphingomyelin/ cholesterol/DOPC TIRFM image (TR-PE in the l d phase) Sphingomyelin/ cholesterol ( l o ) + DOPC ( l d ) From a mixture of DOPC/ sphingomyelin/ cholesterol, domains containing sphingomyelin/ cholesterol ( l o phase) and TR-PE ( l d phase) were enriched in polymer-free regions and partially polymeric bilayer regions, respectively. Okazaki et al. Langmuir 26 , 4126 (2010) 19

Directed phase separation of lipid membranes Possible mechanism Designed polymeric bilayer matrix on a substrate 1) Polymer/ fluid junctions: 4 nm • membrane thickness Low-density No polymer polymer polymer • bending elasticity Introduction of a lipid mixture into the matrix 2) Domain sizes: Lipid mixture of DOPC/ SM/ Chol • restriction by polymeric Spatially controlled phase separation bilayers • Ostwald ripening Lo domains Ld domains DOPC (Ld domain) SM and Chol (Lo domain) 20

Roles of polymeric bilayer matrices Unique feature • Polymerized and fluid bilayers can be integrated at a desired Polymeric and fluid composition by controlling the amount of polymerized bilayers. bilayers are forming a continuous hybrid • Polymeric bilayers can modulate the lateral diffusion of bound membrane molecules and phase separation of l o and l d phases. Roles of polymeric bilayers • Controlling the lateral distribution of molecules • Stabilization of the model membrane 21

Polymeric bilayer edge-induced vesicle fusion (a) Before addition (b) 0.0 min (c) 5.0 min (d) 9.5 min vesicle fusion (e) 10.7 min (f) 12.0 min Pre-formed polymeric bilayers induced the formation of planar bilayers by catalyzing Total internal reflection fluorescence the vesicle fusion process. microscopy (TIR-FM) observation Okazaki et al. Biophys. J. 91 , 1757 (2006) 22

QCM-D results of catalyzed vesicle fusion 10 vesicle Stripe width: 10 μ m DiynePC 0 10 μ m 50 μ m -10 200 μ m Δ f (Hz) -20 SiO 2 -30 -40 rupture -50 4 Stripe width: 50 μ m 3 OR 2 -6 ) Δ D (10 1 bilayer disk 0 -1 -2 0 4 8 12 time (min) Patterned DiynePC bilayers were prepared on QCM-D sensors (SiO 2 coating) with different stripe width for comparing the effect of the density of bilayer edges on the vesicle fusion kinetics. Okazaki et al. Biophys. J. 91, 1757 (2006) 23

QCM-D results of catalyzed vesicle fusion (B) Maximum Δ D in the (A) Time for membrane formation vesicle fusion 5 4 4 3 -6 ) time (min) Δ D peak (10 3 2 2 1 1 0 0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10 -1 ( μ m -1 ) -1 ( μ m -1 ) stripe width stripe width Time for the vesicle fusion and intermediate Δ D maximum values were compared with DiynePC bilayer stripes having different width. Okazaki et al. Biophys. J. 91, 1757 (2006) 24