Dimitris Stroumpoulis

Department of Chemical Engineering - University of California, Santa Barbara

Development of Biomimetic Surfaces by Vesicle Fusion

Introduction

Fibronectin – Integrin System

Cell Surface ReceptorIntegrin family:

Adhesive Ligand:

GRGDSP

PHSRN

10Å

30-40Å

Image adapted from Leahy, D.L. et al., 1996, Cell 84:155

α5β1 α8β1 αvβ1 αvβ3 αvβ5 αvβ6

α2β1 α3β1 α4β1 α7β1

αvβ8 αIIbβ3Integrins Binding RGD (weak)

Intelligent Biomaterials:• Minimize non-specific interactions with ECM• Allow selective interaction with cells

− Mimic ECM (Biomimetics)

Motivation

Design Goals:•Control Microenvironment of Cells•Direct Cell Behavior

Surface Biofunctionalization

Arrange Ligands on Surface:•Accessibility•Conformation•Concentration•Lateral Motion

Planar Bilayer

Polar Headgroup (small)

H/C Tails (bulky)

Properties of Lipid Bilayers:•Bending easier than stretching•Flip flop times are high (102-105 s)•Residence time (104 s)

Peptide Ligand

Langmuir Blodgett-Deposition

Phosphatidylethanolamine

Compression Isotherm1

Surf

ace

pres

sure

Deposition

'Solid'

'Liquid'

2

Area per molecule

GRGDSPPEG120

'Gas’

'Collapse’

• Compress amphiphiles on the air-water interface• Transfer ‘solid’ monolayer onto substrate

Vesicle Fusion

Vesicle Fusion

Planar BilayerVesicle Solution on Surface

Hydrophilic Substrate

Diffusion

Adsorption and deformation

Rupture

Spreading

Rupture propagation

Lateral diffusion

DS1

Substrate

Ellipsometry

Incident BeamReflected Beam

Angle of Incidence

Plane of Incidence

Polarizer

Analyzer

Detector

BirefringenceModulator

)( iri

is

rs

ip

rp

s

p e

E

E

E

E

rr

r δδ −==

δr δi

( )( ) ( )

)Im(2ImRe1

2Im 22 rrr

ry ⋅≈++

=

( )( )( ) z

nnnnn

nnnn

y Δ⋅−−

⋅−+

=Δ 2

22

221

2

22

21

22

212

λπ

rpE

rsE

ipE

isE

Laser Source

Refractive Index of a Single Bilayer

RCOOH (bulk)

RCOOH (Monolayer)

Alkanes (bulk)

Assuming n of one DMPC Bilayer ≅ n of one Monolayer (N=28)

∆z = 40Å

Good Coverage

(From Petrov et al, J. Phys. Chem. 103 (1999) 3417-3424)

Headgroup Area of DMPC 59Å2 ΓM = 3.8 mg/m2

(for ∆ymax = 0.035)

The Model(From “Self Assembly Driven by Hydrophobic Interactions at Alkanethiol Monolayers: Mechanism of

Formation of Hybrid Bilayer Membranes”, J. B. Hubbard, V. Silin, A. L. Plant, Biophysical Chemistry 75 (1998) 163-176 )

2

2

xCD

tC

∂∂⋅=

∂∂1-D Mass Equation:

Mixed Boundary Condition:Surface

SurfaceCK

xCD ⋅=∂∂⋅

Where K: adsorption rate constant of mass (cm/s)Co: bulk concentration of lipids (mg/ml)D: diffusion coefficient of mass close to surface (cm2/s)

)tDKerfc(eCKJ 1/21/2

DtK

oSurface

2

⋅⋅⋅⋅=⋅

Initial Condition:oC0)tC(x, ==

x

General and Limiting Cases

}e{1ΓΓ(t)]

πDt

K21)

DtKerfc(e

KD[

ΓCK

M

2D

t2K

2M

o ⋅+−⋅⋅

−

−⋅=

)e(1ΓΓ(t))

KD

πtD(2

ΓC

MM

o −⋅

−

−⋅=For t/τ >> 1 (Diffusion Limited Case):

)e(1ΓΓ(t) M

oΓ

tCK

M

⋅⋅−

−⋅=For t/τ << 1 (Adsorption Limited Case):

General Solution:

τ = D/K2Characteristic Time of Adsorption:

surfaceMsurface J]

ΓΓ(t)-[1Jβ

dtdΓ

⋅=⋅= Γ(t=0) = 0IC:

Results(Limiting Cases Fittings)

Lipidconcentration

C0, mg ml-1

MaximumSurfacecoverage

Γm, mg cm-2

DiffusioncoefficientD, cm2 s-1

0.4 6.0×10-4 6.6×10-9

0.2 5.5×10-4 1.6×10-8

0.08 6.1×10-4 3.0×10-8

0.02 5.7×10-4 1.2×10-7

Lipidconcentration

C0, mg ml-1

MaximumSurfacecoverage

Γm, mg cm-2

Adsorptionrate constant

K, cm s-1

0.4 3.8×10-4 1.3×10-5

0.2 3.8×10-4 1.1×10-5

0.08 3.8×10-4 1.2×10-5

0.02 3.8×10-4 1.5×10-5

Diffusion Limited Case Adsorption Limited Case

10% mole PA DMPC

Effect of Peptide Amphiphile Presence on the Kinetics

CaseAdsorption

rate constant K, cm s-1

Diffusion coefficientD, cm2 s-1

Diffusion Limited

-------- 7.4×10-8

AdsorptionLimited

1.4×10-5 --------

0.09 mg/ml

O CO

O CO

N C

H

O

C

O

N

H

C

O

N

H

C

O

NH

NH2HN

N

H

C

O

N

H

C

O

CHO O

N

H

C

O

OH

N

H

C

O

OH

G R G D S PC2(C14)2 linker

Kinetics not Interrupted by PA presence

Fluorescence Visualization

1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt(Texas Red® DHPE - Molecular Probes)

DOPC - PA

DOPC – PA 0hrs DOPC – PA 2hrs

2 mm

PA for Cell Adhesion

• Controls the presentation of RGD, a peptide sequence important for cell adhesion, i.e vesicles, bilayers, etc.

• Focused on two peptide amphiphiles with different spacer groups, C2 and polyethylene oxide (PEO).

Tails Spacer Head

O CO

O CO

N C

H

O

CO

NH

CO

NH

CO

N H

N H2H N

NH

CO

NH

CO

CH O O

NH

CO

O H

NH

CO

O H

O C

O

O C

O

N

H

C

O

OO

OC

C

O

N

H

C

O

N

H

C

O

NH

NH2HN

N

H

C

O

N

H

C

O

CHO O

N

H

C

O

OH

N

H

C

O

OH

Experimental Procedure

• Bilayer formation

• Addition of cells

• Vesicle formation

Hydration - Extrusion

100 nm

Glass Substrate

Incubation

Fusion~ 5 nm

• Inertia motion caused by mild microscope stage shaking determines adhesion.

• Scion Imaging software is used to determine the “shape factor.”

Adhesion & Spreading Evaluation

Results EggPC Bilayer

Average Shape Factor- 0.86 (4 hours)No Adhesion

200 μm 200 μm

Results 10% C2-RGD Bilayer

200 μm

Average Shape Factor- 0.84 (3 hours)No Adhesion

200 μm

Results 10 % PEO-RGD Bilayer

Average Shape Factor- 0.52 (4 hours)Adhesion

200 μm 200 μm

Concentration Effect

(2 hours)Adhesion

5% PEO-RGD 10% PEO-RGD

100 μm 100 μm

Surface Concentration Gradients

• Supported bilayers present a physically relevant substrate to study cell-ECM protein interactions

• Active protein sequences can be presented in a control manner using peptide amphiphiles

• There is a drive for the development of multifunctional-multicomponent surfaces

• Further understanding of how specific ligand-integrin interactions modulate cell function is required

• Gradients of membrane bounded ligands can provide a useful tool to study this problem

• Vesicle fusion is the most efficient method to make surface gradients in a membrane environment

Ligand Concentration – Cell Spreading

A

B

C

D

200 μm 200 μm

200 μm 200 μm

Figure: NIH3T3 cells cultured on supported membranes after 4 hours. Membranes in (A) & (B) are 99% EggPC and 1% Texas Red DHPE while in (C) & (D) 94% EggPC, 5% (C16)2-Glu-PEO-GRGDSP and 1% Texas Red DHPE. Pictures (A) and (C) are in optical mode, while (B) and (D) in fluorescent mode on the same surface spot respectively.

Ligand Concentration –Cell Migration

MICROSCOPY RESEARCH AND TECHNIQUE 43:358–368 (1998)

Steps to a Concentration Gradient

Barriers

Patterning

Hydrophilic Substrate

Hydrophilic Substrate

Different ways to make barriers

Materials:• Au, Al, Cr, Ti• Al2O3, TiO2• Fibronectin, BSA• Polymerized Bilayers

Techniques:Standard Photolithography (metals)

• Polymerization• Microcontact Printing• Deep Abrasion (R<<10 nm)

Chemical Nature of Barrier – not topography – Blocks Diffusion

Spread - Neutral and Low PH

Stable - High PH

"Micropattern Formation in Supported Lipid Membranes", Jay T. Groves and Steven G. Boxer, Acc. of Chem. Res., 35, 149-157 (2002).

Chrome Grid

1 mm

200 μm

5 μm EggPC Bilayer

Different ways to pattern

Photolithographic Patterning(Photoactivatable peptides)

Direct Pipetting

Microprinting

Microfluidic Flow

"Micropattern Formation in Supported Lipid Membranes", Jay T. Groves and Steven G. Boxer, Acc. of Chem. Res., 35, 149-157 (2002).

Microfluidic Networks

ConstructionSiO2

SiO2

100 μmSU-8

3 mm

SiO2

Mask

SiO2

Mask

Evaluation

SEM

Testing

Intensity vs Position

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 0.5 1 1.5 2 2.5 3

Position (mm)

Inte

nsity

1 mm

• Ellipsometry was used to study the kinetics of VF on SiO2

• Bilayer formation from vesicles is adsorption limited

• Fluorescence microscopy was used to visualize the membranes

• Peptide accessibility and density are important for cell binding

• Surface composition gradients are useful in studying cellular function modulation induced by ligand-integrin interactions on a biomimetic membrane

• A novel technique was developed to control the microenvironment of cells in 2-D

Conclusions

Acknowledgments

Dr. Haining Zhang PEO chemistry

This work was partially supported by the MRSEC Program of the National Science Foundationunder Award No. DMR00-80034, the National Science Foundation NIRT Award No. CTS-0103516, and the Army Research Office through the Institute for Collaborative Biotechnologies.

Ning Cao Clean-room Processing

Dr. Alejandro Parra Ellipsometry