Cell adhesion to supported peptide-amphiphile bilayer membranes

1. Cell adhesion to supported peptide-amphiphile bilayer membranes Badriprasad Ananthanarayanan Advised by Matthew Tirrell PhD Candidacy exam, August 2004 Faculty Committee: Matthew Tirrell Jacob Israelachvili Samir Mitragotri Luc Jaeger

2. 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).

3. 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).

4. RGD and Integrins • 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 sites on Fibronectin binding to cell-surface integrins Giancotti, FG, et al., Science, 285, 1028 (1999).

5. 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 GRGDSP peptide - headgroup Hydrophobic ‘tail’ section • 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

6. Hydrophilic Substrate 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 incorporating lipids and peptide amphiphiles Vesicle Solution on Surface Vesicle Fusion

7. Lipid Peptide amphiphile Creating Multi-component patterned surfaces Patterned Surfaces Surfaces: - Glass Barriers: - Proteins, e.g. BSA, deposited by microcontact printing Concentration Gradient: - Microfluidic parallel flow - Fabrication of Microchannels Cell adhesion assays

8. Results: Patterned Bilayers Grid-patterned Stamp Patterned bilayer viewed by Fluorescence Microscopy

9. Control glass surfaces for comparison: Results: Cell Adhesion Cells spread to clean glass surfaces but not to fluid lipid bilayers DOPC bilayer viewed by fluorescence and light microscopy

10. Current work • Cell adhesion to bilayers containing peptide-amphiphiles • Fabrication of microchannels for creating patterned surfaces

11. 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?

12. 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).

13. 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).

14. 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).

15. 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).

16. 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).

17. 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).

18. 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).

19. 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

20. 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).

21. 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).

22. 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).

23. FRAP – analysis • Diffusion equation for one species • Solution: Gaussian beam intensity profile, circular spot • Curve fitting gives diffusion constant Axelrod, D et al., Biophys J. 16, 1055 (1976); Ratto TV et al., Biophys J. 83, 3380 (2002).

24. 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).

25. 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).

26. 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).

27. 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