Development of Biomimetic Surfaces by Vesicle Fusionceweb/faculty/tirrell/group_members... ·...
Transcript of Development of Biomimetic Surfaces by Vesicle Fusionceweb/faculty/tirrell/group_members... ·...
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