Composite membranes ofpolymeric and fluid lipid bilayers
K. Morigaki,1,2 T. Okazaki,2 Y. Tatsu,2 and H. Imaishi1
1 Research Center for Environmental Genomics, Kobe University
2 National Institute of Advanced Industrial Science andTechnology (AIST)

1

Membrane functions & biomedical applications
Biomedicalapplications

Membrane functions•Drug development•Diagnostics•Biosensors•Signal transduction•Energy conversion•Immune system•Cell-cell recognition

Measuring and manipulating thefunctions of cellular membranes
at the molecular level

Synthetic model membranes
2Roles of the model membrane systems(biological membrane)ca. 1900

bilayer structure

1960slateral mobility
asymmetry
permeability

From
1980s

3

lipidic

1925

1972

(model membrane)

integral/ peripheral
membrane proteins(fluid mosaic model)
more complex architecture(microdomains, caveolae,membrane skeleton, etc.)

More complex structuresand functions

Langmuir monolayer

lipid vesicles (liposomes)
planar bilayer (BLM)

planar bilayer(substrate supported)
giant vesicles

New model
membranesModel systems of the biological membrane
lipid vesicles(liposomes)

planar lipid bilayer (blacklipid membrane: BLM)

substrate supported
lipid bilayer

1. Mechanical stabilization
2. Surface specific analytical techniques
3. Integration by micro-fabrication
techniques
4Generation of complex model membranes

Supported bilayer

Patterned modelmembrane
polymeric bilayer

natural lipidbilayer

Micro-fabrication & Self-assembly5Fabrication of patterned lipid bilayers(1) Monomer lipid
bilayer (LB/ LS method)

Polymerizable lipid

H2OOOHO P OO+NOOPolymerizationhν(2) Photopolymerization
with a mask

O

R1R1R1h ν (254 nm)

R1R2R2(3) Removal ofmonomer lipids(4) Incorporation ofnew lipid bilayers

6R2R2nPolymerization of lipid bilayers
Synthetic polymerizable lipids: Originally developed for the stabilization ofliposomes for drug delivery applications (Ringsdorf, O’Brien, Chapman, Regen,Tsuchida..)hνΔ

Diacetylene lipid (DiynePC)
R1RO
O

R1

ROOOO
H
O P O
O+NO
OO

R1

R2

R2

R1

R1hν (254 nm)

R1

R2
R2

7

R2R2n

Polymerization in lipid bilayers
UV/visible absorption spectra

Diacetylene lipid (DiynePC)

0.010
0.008+NO
HO P OOAbsorption (AU)OOOOpolymer

0.006
0.004
0.002
0.000

R1R1R1R1hνR1R1R1R1R1R1monomer-0.002
200300400500600

Wavelength (nm)

R2R2R2R2R2

R2R2R2R2R2

Unique features:
1. Topochemical polymerization
2. Conjugated polymer backbone

8Fluorescence spectrafrequency

Patterned polymerization of DiynePC bilayers

0A polymerized Diyne-PC bilayer
after lithographic UV light exposureand removal of monomers. Thescale bar corresponds to 50 μm.

2 4 6height [nm]AFM observation of a lithgraphicallypolymerized DiynePC bilayer. The polymericbilayer consist small domains. The size ofcorrals is 20 μm.

Okazaki et al. Langmuir 25, 345 (2009)9

Fluid bilayers in polymeric bilayer corrals
AB

C

E+D

Fluid lipid bilayers (egg-PC/ TR-PE) can be incorporated into the corrals, and applicationof an electric field induces concentration gradients. The size of corrals is 50 μm.
10Roles of polymeric bilayer matrices
Unique feature
Polymeric and fluidbilayers are forming
a continuous hybrid
membrane

Roles of polymeric bilayers• Controlling the lateral distribution of molecules• Stabilization of the model membrane11Confinement of fluid bilayers in the corrals
ABCD

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 μm.
12Controlling the composition of bilayers
Large UV dose

Small UV dose

Polymeric and fluid bilayers can be integrated as sub-micrometerdomains 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
200frequency

1501005001 x 1 μm0510152025
radius of polymeric domains (nm)30

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

1.5 J/cm2

2.5 J/cm2

4.0 J/cm2

AFM images (1 x 1μm2) 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
5Area fraction from AFM

Polymerized lipid (nm)

1.0

4
32
1
00

12
34
562
UV irradiation dose (J/cm )7Film thickness after the 0.1M SDStreatment (ellipsometry)

0.8
0.6
0.4
0.2
0.0
0.0

0.2
0.4
0.6
0.8
1.0
Area fraction from ellipsometry

The area fractions of polymeric bilayers
from AFM images were plotted versus
those from the ellipsometry andfluorescence 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

(A) Polymer(B) Fluid

252

50

303

Fluorescence microscopy images of apatterned hybrid SPB with spatially varied
degree of polymerization ( UV doses shown in
J/cm2).
(A) Fluorescence from polymeric DiynePC. (B)Fluorescence from fluid bilayers. The scalebars correspond to 100 μm.

Fluorescence intensity (normalized)

Composition of polymerized and fluid bilayers
1.0
0.8
0.6
0.4
0.2
0.001234
Polymerized lipid (nm)5

The amount of fluid bilayers(normalized to full coverage) andpolymeric bilayers (Determined byfluorescence microscopy)

The amount of fluid bilayers incorporated changed linearly with the amountof polymeric bilayers, suggesting the formation of a composite membrane.
Okazaki et al. Langmuir 25, 345 (2009)17

Obstructed diffusion by polymeric membranes
Relative diffusion coefficient (D*)(a) 1.5 J/cm21.0
Fitting to thefree-area model(b) 2.5 J/cm2a0.5b(c) 4.0 J/cm20.0

c0.0

0.2
0.4
0.6
0.8
Area fraction of obstacle (c)1.0Diffusion coefficient of TR-PE was plotted as a function of the amount ofpolymeric bilayer in the composite membrane. Diffusion coefficients weredetermined by the fluorescence recovery after photobleaching (FRAP) method.
18Okazaki et al. Langmuir 25, 345 (2009)Phase separation of lipid membranes
Sphingomyelin/ cholesterol/DOPCTIRFM image (TR-PE in the ld phase)

Sphingomyelin/ cholesterol (lo) + DOPC (ld)

From a mixture of DOPC/ sphingomyelin/ cholesterol, domains containing
sphingomyelin/ cholesterol (lo phase) and TR-PE (ld phase) were enriched inpolymer-free regions and partially polymeric bilayer regions, respectively.
Okazaki et al. Langmuir 26, 4126 (2010)19

Directed phase separation of lipid membranes
Designed polymeric bilayer matrix on a substrate

4 nm

No polymer

Low-density polymer
polymerIntroduction of a lipid mixture into the matrix

Lipid mixture of DOPC/ SM/ Chol
Spatially controlled phase separationLo domains

Ld domains

DOPC (Ld domain)
SM and Chol (Lo domain)

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Possible mechanism
1) Polymer/ fluid junctions:• membrane thickness• bending elasticity
2) Domain sizes:• restriction by polymericbilayers• Ostwald ripening

Roles of polymeric bilayer matrices
Unique feature• Polymerized and fluid bilayers can be integrated at a desired
Polymericand fluid bilayers.
composition by controlling the amountof polymerized
bilayers are forming
continuous
hybrid of bound• Polymeric bilayers can modulatea thelateral diffusion
molecules and phase separation membraneof lo and ld phases.

Roles of polymeric bilayers• Controlling the lateral distribution of molecules• Stabilization of the model membrane

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Polymeric bilayer edge-induced vesicle fusion

vesiclefusionPre-formed polymeric bilayers
induced the formation ofplanar bilayers by catalyzing
the vesicle fusion process.

(a) Before addition(b) 0.0 min(c) 5.0 min(d) 9.5 min(e) 10.7 min(f) 12.0 min

Total internal reflection fluorescencemicroscopy (TIR-FM) observation
Okazaki et al. Biophys. J. 91, 1757 (2006)

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QCM-D results of catalyzed vesicle fusion
10Stripe width: 10μmvesicle

DiynePC
010μm

Δf (Hz)-1050μm

200μm

-20

SiO2-30-40

rupture-50
4Stripe width: 50μm

OR2-6ΔD (10 )31bilayer disk

0-1-204812time (min)Patterned DiynePC bilayers were prepared on QCM-D sensors (SiO2 coating) withdifferent stripe width for comparing the effect of the density of bilayer edges on thevesicle fusion kinetics.
23Okazaki et al. Biophys. J. 91, 1757 (2006)

QCM-D results of catalyzed vesicle fusion

5443-6

ΔD peak (10 )(B) Maximum ΔD in thevesicle fusion

time (min)(A) Time for membrane
formation322
1

100.00 0.02 0.04 0.06 0.08 0.10
-1
-1
stripe width (μm )0.00 0.02 0.04 0.06 0.08 0.10
-1
-1
stripe width (μm )Time for the vesicle fusion and intermediate ΔD maximum values were comparedwith DiynePC bilayer stripes having different width.
Okazaki et al. Biophys. J. 91, 1757 (2006)
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QCM-D results of partially polymeric bilayers

2.0J/cm24.0J/cm21.0J/cm2Δf (Hz)100-10-20-30-40-50
432
10-1-20.5J/cm2-6ΔD (10 )SiO2Changes of the resonant frequency (Δf)
and dissipation (ΔD) measured by theQCM-D during the vesicle fusion on
SiO2 (black) and polymerized DiynePC
surfaces. The UV irradiation dose forthe photopolymerization was 4.0 J/cm2(light-blue), 2.0 J/cm2 (blue), 1.0 J/cm2(green) and 0.5 J/cm2 (red), respectively.

Positive Δf and negative ΔD:
Effects of bound watermolecules at bilayer edges?
02

4
6
8time (min)1012
Okazaki et al. Langmuir 25, 345 (2009)25

Roles of polymeric bilayer matrices
Unique feature• Formation of planar bilayers was enhanced at the boundaries
Polymeric and fluidof polymeric bilayers.
bilayers are forming
a continuoushybrid• Defects formed in planar bilayers
in polymer-freeregions,membrane
whereas partially polymeric regions
accumulated more lipid.

Roles of polymeric bilayers• Controlling the lateral distribution of molecules• Stabilization of the model membrane

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Summary of the results•

Polymeric and fluid bilayers can be integrated as a continuouscomposite membrane.

•Polymeric bilayers act as obstacles for lateral migration of
membrane-bound molecules.

•Polymeric bilayers can affect the spatial distribution ofmolecules by inducing guided phase separation of lipids.

•Junctions of polymeric and fluid bilayers play important rolesin the formation and stability of membranes.

In short:
Fluid bilayers are a confined 2D soft matter witha strong coupling with the boundaries
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Acknowledgement
People:AIST
Takashi Okazaki
Saori Mori
Yoshiro Tatsu
Noboru YumotoTakehiko Inaba
Takashi Irie
Junji Nishii

Shigeki Kimura
Yoshihiro Nakajima
Takahisa Taguchi

Kobe University
Hiromasa Imaishi
Institute for Molecular Science
Ryugo Tero
Tsuneo Urisu

Financial support:•Promotion Budget for Science and Technology (MEXT)•Grant-in-Aid for Scientific research (JSPS)•Sekisui Chemical Grant Program•Program for Promotion of Basic Research Activities for Innovative Biosciences(PROBRAIN)
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