Cell adhesion to supported peptide-amphiphile bilayer

membranesBadriprasad 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 scaffoldsCells 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 othersprovide a structural framework and biochemicalsignals that control cellular function, e.g. adhesion,growth, differentiation, etc.

Creating biomaterials which reproduce these interactionsmay allow us to direct cell adhesion

Tirrell, M et al., Surface Science, 500, 61 (2000).

• Fibronectin is one of the adhesion-promoting proteins in the ECM

• Fibronectin binds to cell-surface receptors known as integrins, trans-membrane 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 and Integrins

RGD sites on Fibronectinbinding to cell-surface integrins

Giancotti, FG, et al., Science, 285, 1028 (1999).

Peptide biomaterials: peptide-amphiphiles

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

C

HOO

N

H

C

O

OH

N

H

C

O

OH

Hydrophobic ‘tail’ section

Peptide amphiphiles

• 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 self-assembly

• 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

GRGDSP peptide - headgroup

Self-assembly: Vesicle Fusion

Vesicle Fusion

Vesicle Solution on Surface

Hydrophilic Substrate

Vesicle incorporating lipids and peptide amphiphiles

• 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

Patterned Surfaces

Surfaces: - Glass Barriers: - Proteins, e.g. BSA,

deposited by microcontact printing

Concentration Gradient: - Microfluidic parallel flow - Fabrication of Microchannels

Lipid

Peptide amphiphile

Cell adhesion assays

Creating Multi-component patterned surfaces

Results: Patterned Bilayers

Grid-patterned Stamp

Patterned bilayer viewed by Fluorescence

Microscopy

DOPC bilayer viewed by fluorescence and light microscopy

Results: Cell Adhesion

Cells spread to clean glass surfaces but not to fluid lipid bilayers

Control glass surfaces for comparison:

Current work

Cell adhesion to bilayers containing peptide-amphiphiles

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 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

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

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 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 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 Controlling fluidity in SLBs Characterizing fluidity – FRAP Cell adhesion assays SLB microstructure – formation of domains

SLB – controlling fluidity

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

Quenching mixed-lipid bilayers below the melting temperature 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

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

Diffusion equation for one species

Solution: Gaussian beam intensity profile, circular spot

Curve fitting gives diffusion constant

),(),( 2 trCD

t

trC

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

Centrifugal detachment assay Sample plate spun in

centrifuge, adherent cells counted before and after

Low detachment forces applied Hydrodynamic flow

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