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Cell adhesion to
supported peptideamphiphile bilayer
membranes
Badriprasad Ananthanarayanan
Advised by
Matthew Tirrell
PhD Candidacy exam, August 2004
Faculty Committee:
Matthew Tirrell
Jacob Israelachvili
Samir Mitragotri
Luc Jaeger
Introduction

Biomaterials
Surface functionalization for increased compatibility and
safety
Examples
 Implant materials, e.g. Vascular grafts

Seeding with endothelial cells improves
graft performance

Tissue engineering scaffolds
Cells require many signals from matrix to enable
proliferation and tissue regrowth
Tirrell, M et al., Surface Science, 500, 61 (2000).
Biomimetics

Engineering biological recognition to create ‘biomimetic’
materials

Extra-Cellular Matrix
Proteins in the ECM e.g. fibronectin and others
provide a structural framework and biochemical
signals that control cellular function, e.g. adhesion,
growth, differentiation, etc.
Creating biomaterials which reproduce these interactions
may allow us to direct cell adhesion
Tirrell, M et al., Surface Science, 500, 61 (2000).
RGD and Integrins
• Fibronectin is one of the adhesion-promoting proteins in the ECM
• Fibronectin binds to cell-surface receptors known as integrins, transmembrane proteins which regulate a number of cellular processes
• The binding site for many integrins in fibronectin is the loop containing
the peptide sequence Arg-Gly-Asp (RGD)
RGD sites on Fibronectin
binding to cell-surface integrins
Giancotti, FG, et al., Science, 285, 1028 (1999).
Peptide biomaterials:
peptide-amphiphiles
•
Short peptides incorporating the RGD sequence can bind integrins and
promote cell adhesion, similar to fibronectin
•
Using peptides may offer advantages over proteins in terms of convenience,
selectivity, and presentation on surfaces
Peptide amphiphiles
NH2
HN
NH
HO
O
O
C
O
C
N
H
O
C
C
O
H
O
N
C
N
C
H
O
H
O
N
C
O
N
C
H
O
H
O
N
C
N
C
H
O
OH
OH
C
O
Hydrophobic ‘tail’ section
GRGDSP peptide - headgroup
• Peptide headgroups covalently linked to a hydrophobic ‘tail’ segment
• Hydrophobic-force driven self-assembly into micelles, vesicles, bilayers, etc.
allows us to easily deposit functional molecules on surfaces using selfassembly
Self-assembly: Vesicle Fusion
• Vesicles are formed from a solution of amphiphiles
• When exposed to a hydrophilic surface, vesicles
rupture and form bilayer fragments which fuse to form
a continuous bilayer on the surface
• Clean hydrophobic surfaces are essential for fusion,
smaller vesicles are more fusogenic
Vesicle Solution on Surface
Vesicle
Fusion
Hydrophilic Substrate
Patterned Surfaces
Creating Multi-component
patterned surfaces
Lipid
Peptide amphiphile
Surfaces:
- Glass
Barriers:
- Proteins, e.g. BSA,
deposited by
microcontact printing
Concentration Gradient:
- Microfluidic parallel flow
- Fabrication of
Microchannels
Cell adhesion assays
Results: Patterned Bilayers
Grid-patterned
Stamp
Patterned bilayer
viewed by Fluorescence
Microscopy
Results: Cell Adhesion
Cells spread to clean glass surfaces but not to fluid lipid bilayers
DOPC bilayer viewed by fluorescence and light microscopy
Control glass surfaces for comparison:
Current work


Cell adhesion to bilayers containing peptideamphiphiles
Fabrication of microchannels for creating
patterned surfaces
Effect of Membrane Fluidity on
Cell Adhesion

SLBs used in our research as a platform for
incorporating adhesion-promoting ligands

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Ease of fabrication by vesicle fusion
Inert background: cells show no adhesion to fluid
lipid bilayers
Retains lateral mobility of membrane components
and hence a better mimic of cell membrane
Fluidity of SLBs has been used for various
purposes



Creating micropatterned surfaces
Biosensors, etc.
Does the fluidity have an effect on cell adhesion?
Membrane fluidity in nature

Fluid Mosaic model of membranes – proteins and
lipids have varying degrees of lateral fluidity

Lateral mobility of membrane proteins is an
essential step in many signal transduction
pathways, e.g. action of soluble hormones,
immune recognition, growth, etc.
Jacobson, K et al., Science 268, 1441 (1995).
Example: Immune Recognition



T-cell activation is a critical step in the immune response
T-cell activation requires sustained engagement of T-cell
receptors by ligands through the ‘immunological synapse’
Formation of this structure involves many receptor-ligand pairs
and their transport within the membrane
Groves, JT et al., J. Immunol. Meth. 278, 19 (2003).
Influence of Ligand Mobility


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T-cell receptor CD2 and its counter-receptor CD58 (LFA-3) –
one of the receptor-ligand pairs involved in T-cell signalling
CD58 found in two forms: lipid-anchored (GPI) and
transmembrane (TM)
lipid-anchored form was mobile, TM form immobile
Adhesion of T-cells to GPI-anchored form at lower densities,
and adhesion strength also higher
Chan, P-Y et al., J. Cell. Bio. 115, 245 (1991).
Cell adhesion: RGD and integrins
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Integrins association with
ECM is essential for cell
adhesion and motility
Integrins cluster as they
bind, enabling assembly of
their cytoplasmic domains
which initiates actin stress
fiber formation
This results in more integrin
clustering, binding and
finally, formation of focal
contacts essential for stable
adhesion
Ruoslahti, E et al., Science 238, 491 (1987); Giancotti FG et al., Science 285, 1028 (1999).
Effect of RGD clustering

The effect of RGD surface density is well known

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Average ligand spacing of 440 nm for spreading, 140 nm for focal
contacts
Some evidence that clustering of ligands facilitates cell adhesion

(RGD)n-BSA conjugates show equivalent adhesion at much lower RGD
densities for higher values of n

Synthetic polymer-linked RGD clusters show more efficient adhesion
and well-formed stress fibers for nine-member clusters
Danilov YN et al., Exp. Cell Res. 182, 186 (1989).
Effect of RGD clustering
• There is a definite effect of nanoscale clustering of ligands on cell
adhesion
Maheshwari G et al., J. Cell Sci. 113, 1677 (2000).
Simulation of RGD clustering

Single-state model – clustering of ligands does not
change binding affinity KD


No effect observed on ligand clustering other than receptor
clustering
Two-state model – ligand clustering causes increase
in KD – represents activation of receptor in vivo

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Significantly higher number of receptors bound, especially
at low average ligand density
This translates into stronger adhesion and better assembly
of focal contacts
Irvine, DJ et al., Biophys. J. 82, 120 (2002).
Effect of bilayer fluidity


Spatial organization of ligand has a great
effect on cell adhesion, hence fluidity of SLB
may have an effect
Experimental plan
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Controlling fluidity in SLBs
Characterizing fluidity – FRAP
Cell adhesion assays
SLB microstructure – formation of domains
SLB – controlling fluidity
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Polymerizable Lipid tails

Diacetylenic moieties in lipid tails – can be polymerized by
UV irradiation

Polymerizable tails can be conjugated to RGD, or lipids
with polymerizable tails can be used as a background
Control fluidity by varying the degree of polymerization as
well as the concentration of polymerizable molecules

Tu, RS, PhD thesis, UCSB (2004).
SLB – controlling fluidity
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Quenching mixed-lipid bilayers below the
melting temperature
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e.g. mixed DLPC/DSPC vesicles quenched from
700C to room temperature
Results in formation of small lipid domains
These domains act as obstacles to lateral
diffusion in the bilayer
When solid-phase area fraction is very high,
diffusion of fluid-phase molecules goes to zero
Ratto TV et al., Biophys J. 83, 3380 (2002).
Characterizing Fluidity – FRAP
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Fluorescence Recovery After Photobleaching
Fluorescent molecules bleached by high-intensity light source or
laser pulse
The same light source, highly attenuated, is used to monitor
recovery of fluorescence due to diffusion of fluorescent molecules
into the bleached area
Spot bleaching or Pattern Bleaching
Curve fitting gives diffusion constant and mobile fraction
Groves, JT et al., Langmuir 17, 5129 (2001).
FRAP – analysis

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Diffusion equation for
one species
Solution: Gaussian
beam intensity profile,
circular spot
C (r , t )
 D 2 C (r , t )
t
Curve fitting gives
diffusion constant
Axelrod, D et al., Biophys J. 16, 1055 (1976); Ratto TV et al., Biophys J. 83, 3380 (2002).
FRAP – instrument setup
• Light source: High-power lamp or laser
• Electromechanical shutter system used to switch between high-intensity beam
and fluorescence observation light
• PMT vs. Camera – camera allows spatial resolution of intensity, and hence we
can monitor background fluorescence recovery, other transport processes
• Data analysis by image-analysis software
Meyvis, TLK, et al., Pharm. Res. 16, 1153 (1999).
Cell adhesion assays
Determining adhesion
strength
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Centrifugal detachment assay
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Sample plate spun in
centrifuge, adherent cells
counted before and after
Low detachment forces applied
Hydrodynamic flow
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Shear stress applied due to
flow
Many configurations possible
Detachment force may depend
on cell morphology
Garcia, AJ et al., Cell Biochem. Biophys. 39, 61 (2003).
Cell adhesion assays
Detect extent of cytoskeletal
organization and focal
adhesion assembly
 Staining of actin filaments
to visualize stress fiber
formation
 Population of cells that
show well-formed stress
fibers can be visually
determined
Maheshwari, G et al., J. Cell. Sci. 113, 1677 (2000).
Conclusions




Constructing supported bilayer membranes
incorporating peptide-amphiphiles for cell adhesion
Creating micropatterned surfaces for displaying
spatially varied ligand concentrations
Effect of bilayer fluidity on cell adhesion strength
and focal adhesion assembly
Design of efficient biomimetic surfaces for analytical
or biomedical applications