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

1

Composite membranes of polymeric 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 and

Technology (AIST)

2

Membrane functions

• Signal transduction
• Energy conversion
• Immune system
• Cell-cell recognition

Biomedical applications

• Drug development
• Diagnostics
• Biosensors

Synthetic model membranes Membrane functions & biomedical applications

Measuring and manipulating the functions of cellular membranes at the molecular level

3

Roles of the model membrane systems

More complex structures and functions New model membranes

(biological membrane) lipidic bilayer structure lateral mobility asymmetry permeability integral/ peripheral membrane proteins (fluid mosaic model) (model membrane) Langmuir monolayer lipid vesicles (liposomes) planar bilayer (BLM) planar bilayer (substrate supported)

• ca. 1900

1925 1960s 1972 From 1980s

more complex architecture (microdomains, caveolae, membrane skeleton, etc.) giant vesicles

4

lipid vesicles (liposomes) planar lipid bilayer (black lipid membrane: BLM) substrate supported lipid bilayer

Model systems of the biological membrane

• 1. Mechanical stabilization
• 2. Surface specific analytical techniques
• 3. Integration by micro-fabrication

techniques

5

Generation of complex model membranes

Supported bilayer

Micro-fabrication & Self-assembly

Patterned model membrane

polymeric bilayer natural lipid bilayer

6

Fabrication of patterned lipid bilayers

hν

(1) Monomer lipid bilayer (LB/ LS method) (2) Photopolymerization with a mask (3) Removal of monomer lipids (4) Incorporation of new lipid bilayers

H2O

O O O O H O P O O O N

+

R1 R2 R1 R2 R1 R1 R2 R2 n hν (254 nm)

Polymerizable lipid Polymerization

7

Polymerization of lipid bilayers

R R1 R2 R1 O R2 O R O O

O O O O H O P O O O N

+

Diacetylene lipid (DiynePC)

R1 R2 R1 R2 R1 R1 R2 R2 n hν (254 nm)

Synthetic polymerizable lipids: Originally developed for the stabilization of liposomes for drug delivery applications (Ringsdorf, O’Brien, Chapman, Regen, Tsuchida..) hν Δ

8

Polymerization in lipid bilayers

O O O O H O P O O O N

+

Diacetylene lipid (DiynePC)

R2 R2 R2 R2 R2 R1 R1 R1 R1 R1 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2

hν

UV/visible absorption spectra

200 300 400 500 600

• 0.002

0.000 0.002 0.004 0.006 0.008 0.010

Absorption (AU) Wavelength (nm)

monomer polymer

Fluorescence spectra

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

9

Patterned polymerization of DiynePC bilayers

A polymerized Diyne-PC bilayer after lithographic UV light exposure and removal of monomers. The scale bar corresponds to 50 μm.

2 4 6 frequency height [nm]

AFM observation of a lithgraphically polymerized DiynePC bilayer. The polymeric bilayer consist small domains. The size of corrals is 20 μm.

Okazaki et al. Langmuir 25, 345 (2009)

10

Fluid bilayers in polymeric bilayer corrals

Fluid lipid bilayers (egg-PC/ TR-PE) can be incorporated into the corrals, and application

• f an electric field induces concentration gradients. The size of corrals is 50 μm.

A B

• +

E

C D

11

Polymeric and fluid bilayers are forming a continuous hybrid membrane

Roles of polymeric bilayer matrices

Unique feature Roles of polymeric bilayers

• Controlling the lateral distribution of molecules
• Stabilization of the model membrane

12

Confinement of fluid bilayers in the corrals

The bleached corral became homogeneously dark, indicating that lipid molecules are diffusing laterally within the corral. The scale bar corresponds to 50 μm.

A B C D

4 min after the photobleach

13

Large UV dose Small UV dose

JP-Patent 4,150,793 (2008) Morigaki et al. Langmuir 20, 7729 (2004)

Polymeric and fluid bilayers can be integrated as sub-micrometer domains by modulating the degree of polymerization.

Controlling the composition of bilayers

14

5 10 15 20 25 30 50 100 150 200 frequency radius of polymeric domains (nm)

Size distribution of polymeric bilayer domains

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. 1 x 1 μm

Okazaki et al. Langmuir 25, 345 (2009)

15

1.5 J/cm2 4.0 J/cm2 2.5 J/cm2

Polymeric bilayers with different UV doses

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)

16

Film thickness after the 0.1M SDS treatment (ellipsometry)

The amount of polymerized bilayers can be controlled by changing the applied UV dose for polymerization

The amount of polymerized bilayers

The area fractions of polymeric bilayers from AFM images were plotted versus those from the ellipsometry and fluorescence microscopy.

1 2 3 4 5 6 7 1 2 3 4 5 Polymerized lipid (nm) UV irradiation dose (J/cm

2)

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Area fraction from AFM Area fraction from ellipsometry

Okazaki et al. Langmuir 25, 345 (2009)

17

The amount of fluid bilayers incorporated changed linearly with the amount

• f polymeric bilayers, suggesting the formation of a composite membrane.

Composition of polymerized and fluid bilayers

The amount of fluid bilayers (normalized to full coverage) and polymeric bilayers (Determined by fluorescence microscopy)

1 2 3 4 5 0.0 0.2 0.4 0.6 0.8 1.0 Fluorescence intensity (normalized) Polymerized lipid (nm)

Fluorescence microscopy images of a patterned 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 scale bars correspond to 100 μm.

2 5 3 2 5 3

(A) Polymer (B) Fluid

Okazaki et al. Langmuir 25, 345 (2009)

18

Obstructed diffusion by polymeric membranes

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.

(a) 1.5 J/cm2 (c) 4.0 J/cm2 (b) 2.5 J/cm2

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.5 1.0 Relative diffusion coefficient (D

*)

Area fraction of obstacle (c)

Fitting to the free-area model a b c

Okazaki et al. Langmuir 25, 345 (2009)

19

Phase separation of lipid membranes

From a mixture of DOPC/ sphingomyelin/ cholesterol, domains containing sphingomyelin/ cholesterol (lo phase) and TR-PE (ld phase) were enriched in polymer-free regions and partially polymeric bilayer regions, respectively.

Sphingomyelin/ cholesterol/DOPC Sphingomyelin/ cholesterol (lo) + DOPC (ld)

TIRFM image (TR-PE in the ld phase)

Okazaki et al. Langmuir 26, 4126 (2010)

20

polymer Low-density polymer No polymer Designed polymeric bilayer matrix on a substrate Lipid mixture of DOPC/ SM/ Chol Introduction of a lipid mixture into the matrix Ld domains Lo domains Spatially controlled phase separation

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

Directed phase separation of lipid membranes

1) Polymer/ fluid junctions:

• membrane thickness
• bending elasticity

2) Domain sizes:

• restriction by polymeric

bilayers

• Ostwald ripening

Possible mechanism

21

Polymeric and fluid bilayers are forming a continuous hybrid membrane

Roles of polymeric bilayer matrices

Unique feature Roles of polymeric bilayers

• Polymerized and fluid bilayers can be integrated at a desired

composition by controlling the amount of polymerized bilayers.

• Polymeric bilayers can modulate the lateral diffusion of bound

molecules and phase separation of lo and ld phases.

• Controlling the lateral distribution of molecules
• Stabilization of the model membrane

22 Okazaki et al. Biophys. J. 91, 1757 (2006)

Polymeric bilayer edge-induced vesicle fusion

Total internal reflection fluorescence microscopy (TIR-FM) observation

Pre-formed polymeric bilayers induced the formation of planar bilayers by catalyzing the vesicle fusion process.

vesicle fusion

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

23

QCM-D results of catalyzed vesicle fusion

Okazaki et al. Biophys. J. 91, 1757 (2006)

Patterned DiynePC bilayers were prepared on QCM-D sensors (SiO2 coating) with different stripe width for comparing the effect of the density of bilayer edges on the vesicle fusion kinetics.

Stripe width: 10μm Stripe width: 50μm

• 50
• 40
• 30
• 20
• 10

10 4 8 12

• 2
• 1

1 2 3 4

Δf (Hz) ΔD (10

• 6)

time (min)

SiO2 200μm 50μm 10μm DiynePC

vesicle OR rupture bilayer disk

24

Time for the vesicle fusion and intermediate ΔD maximum values were compared with DiynePC bilayer stripes having different width.

Okazaki et al. Biophys. J. 91, 1757 (2006)

0.00 0.02 0.04 0.06 0.08 0.10 1 2 3 4 5 time (min) stripe width

• 1 (μm
• 1)

0.00 0.02 0.04 0.06 0.08 0.10 1 2 3 4

ΔD peak (10

• 6)

stripe width

• 1 (μm
• 1)

(A) Time for membrane formation (B) Maximum ΔD in the vesicle fusion

QCM-D results of catalyzed vesicle fusion

25

Changes of the resonant frequency (Δf) and dissipation (ΔD) measured by the QCM-D during the vesicle fusion on SiO2 (black) and polymerized DiynePC

• surfaces. The UV irradiation dose for

the 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.

Okazaki et al. Langmuir 25, 345 (2009)

QCM-D results of partially polymeric bilayers

Positive Δf and negative ΔD: Effects of bound water molecules at bilayer edges?

2 4 6 8 10 12

• 2
• 1

1 2 3 4

• 50
• 40
• 30
• 20
• 10

10

ΔD (10

• 6)

time (min)

Δf (Hz)

0.5J/cm2 1.0J/cm2 4.0J/cm2 2.0J/cm2 SiO2

26

• Controlling the lateral distribution of molecules
• Stabilization of the model membrane

Polymeric and fluid bilayers are forming a continuous hybrid membrane

Roles of polymeric bilayer matrices

Unique feature Roles of polymeric bilayers

• Formation of planar bilayers was enhanced at the boundaries
• f polymeric bilayers.
• Defects formed in planar bilayers in polymer-free regions,

whereas partially polymeric regions accumulated more lipid.

27

• Polymeric and fluid bilayers can be integrated as a continuous

composite membrane.

• Polymeric bilayers act as obstacles for lateral migration of

membrane-bound molecules.

• Polymeric bilayers can affect the spatial distribution of

molecules by inducing guided phase separation of lipids.

• Junctions of polymeric and fluid bilayers play important roles

in the formation and stability of membranes.

Summary of the results

In short: Fluid bilayers are a confined 2D soft matter with a strong coupling with the boundaries

28

Acknowledgement Acknowledgement

People:

AIST Takashi Okazaki Takehiko Inaba Shigeki Kimura Saori Mori Takashi Irie Yoshihiro Nakajima Yoshiro Tatsu Junji Nishii Takahisa Taguchi Noboru Yumoto 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)