Development of Biomimetic Surfaces by Vesicle Fusion

Transcription

1 Development of Biomimetic Surfaces by Vesicle Fusion Dimitris Stroumpoulis Department of hemical Engineering - University of alifornia, Santa Barbara

2 Introduction

3 Fibronectin Integrin System ell Surface Receptor Integrin family: Adhesive Ligand: GRGDSP 10Å 30-40Å PSR Integrins Binding RGD α 5 β 1 α 8 β 1 α v β 1 α v β 3 α v β 5 α v β 6 α 2 β 1 α 3 β 1 α 4 β 1 α 7 β 1 (weak) α v β 8 α IIb β 3 Image adapted from Leahy, D.L. et al., 1996, ell 84:155

4 Intelligent Biomaterials: Minimize non-specific interactions with EM Allow selective interaction with cells Mimic EM (Biomimetics) Motivation Design Goals: ontrol Microenvironment of ells Direct ell Behavior

5 Surface Biofunctionalization Arrange Ligands on Surface: Accessibility onformation oncentration Lateral Motion Peptide Ligand Polar eadgroup (small) / Tails (bulky) Planar Bilayer Properties of Lipid Bilayers: Bending easier than stretching Flip flop times are high ( s) Residence time (10 4 s)

6 Langmuir Blodgett-Deposition ompress amphiphiles on the air-water interface Transfer solid monolayer onto substrate 'ollapse 2 Deposition Surface pressure 'Solid' 'Liquid' 'Gas Phosphatidylethanolamine GRGDSP PEG120 1 Area per molecule ompression Isotherm

7 DS1 Vesicle Fusion Vesicle Solution on Surface Planar Bilayer Vesicle Fusion ydrophilic Substrate Diffusion Adsorption and deformation Rupture Spreading Rupture propagation Lateral diffusion

8 Ellipsometry Angle of Incidence Reflected Beam Incident Beam Detector Laser Source r E p i E p Polarizer Analyzer r E s δ r δ i i E s Birefringence Modulator Substrate Plane of Incidence y = 2 Im() r 2 Im( r) Re ( 2 2 n n ) () r + Im() r 2 2 2π n1 + n2 Δy = 2 λ n 1 2 ( 2 2 )( 2 2 n n n n ) 1 2 Δz r = r r p s = E E E E r p i p r s i s e i δ δ ) ( r i

9 Refractive Index of a Single Bilayer R (bulk) R (Monolayer) Alkanes (bulk) (From Petrov et al, J. Phys. hem. 103 (1999) ) Assuming n of one DMP Bilayer n of one Monolayer (=28) z = 40Å (for y max = 0.035) Good overage eadgroup Area of DMP 59Å 2 Γ M = 3.8 mg/m 2

10 The Model (From Self Assembly Driven by ydrophobic Interactions at Alkanethiol Monolayers: Mechanism of Formation of ybrid Bilayer Membranes, J. B. ubbard, V. Silin, A. L. Plant, Biophysical hemistry 75 (1998) ) x 1-D Mass Equation: t = D 2 2 x Initial ondition: (x, t = 0) = o Mixed Boundary ondition: D x Surface = K Surface Where K: adsorption rate constant of mass (cm/s) o : bulk concentration of lipids (mg/ml) D: diffusion coefficient of mass close to surface (cm 2 /s) J Surface = K o e 2 K t D K erfc( D 1/2 t 1/2 )

11 General and Limiting ases dγ dt Γ(t) = β J = [1- ] J surface I: Γ(t=0) = 0 Γ M surface General Solution: Γ(t) = Γ M {1 e K Γ M o D [ (e 2 K K 2 t D erfc 2 K t 2 1) + D K Dt π ] } haracteristic Time of Adsorption: τ = D/K 2 For t/τ >> 1 (Diffusion Limited ase): Γ(t) = Γ M (1 e Γ o M (2 D t D ) π K ) For t/τ << 1 (Adsorption Limited ase): Γ(t) = Γ M (1 e K Γ o M t )

12 Results (Limiting ases Fittings) Diffusion Limited ase Adsorption Limited ase Lipid concentration 0, mg ml -1 Maximum Surface coverage Γ m, mg cm -2 Diffusion coefficient D, cm 2 s -1 Lipid concentration 0, mg ml -1 Maximum Surface coverage Γ m, mg cm -2 Adsorption rate constant K, cm s

13 Effect of Peptide Amphiphile Presence on the Kinetics 2 ( 14 ) 2 linker 2 G R G D S P 0.09 mg/ml 10% mole PA DMP ase Diffusion Limited Adsorption Limited Adsorption rate constant K, cm s Diffusion coefficient D, cm 2 s Kinetics not Interrupted by PA presence

14 Fluorescence Visualization DP - PA 1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt (Texas Red DPE - Molecular Probes) 2 mm DP PA 0hrs DP PA 2hrs

15 PA for ell Adhesion ontrols 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, 2 and polyethylene oxide (PE). Tails Spacer ead 2 2

16 Experimental Procedure Vesicle formation 100 nm ydration - Extrusion Bilayer formation Fusion ~ 5 nm Glass Substrate Addition of cells Incubation

17 Adhesion & Spreading Evaluation Inertia motion caused by mild microscope stage shaking determines adhesion. Scion Imaging software is used to determine the shape factor.

18 Results EggP Bilayer 200 μm 200 μm Average Shape Factor (4 hours) o Adhesion

19 Results 10% 2 -RGD Bilayer 200 μm 200 μm Average Shape Factor (3 hours) o Adhesion

20 Results 10 % PE-RGD Bilayer 200 μm 200 μm Average Shape Factor (4 hours) Adhesion

21 oncentration Effect 100 μm 100 μm 5% PE-RGD 10% PE-RGD (2 hours) Adhesion

22 Surface oncentration Gradients Supported bilayers present a physically relevant substrate to study cell-em protein interactions Active protein sequences can be presented in a control manner using peptide amphiphiles There is a drive for the development of multifunctionalmulticomponent 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

23 Ligand oncentration ell Spreading A 200 μm 200 μm B D 200 μm 200 μm Figure: I3T3 cells cultured on supported membranes after 4 hours. Membranes in (A) & (B) are 99% EggP and 1% Texas Red DPE while in () & (D) 94% EggP, 5% (16)2-Glu-PE-GRGDSP and 1% Texas Red DPE. Pictures (A) and () are in optical mode, while (B) and (D) in fluorescent mode on the same surface spot respectively.

24 Ligand oncentration ell Migration MIRSPY RESEAR AD TEIQUE 43: (1998)

25 Steps to a oncentration Gradient Barriers ydrophilic Substrate Patterning ydrophilic Substrate

26 Different ways to make barriers Materials: Au, Al, r, Ti Al 2 3, Ti 2 Fibronectin, BSA Polymerized Bilayers hemical ature of Barrier not topography Blocks Diffusion Techniques: Standard Photolithography (metals) Polymerization Microcontact Printing Spread - eutral and Low P Deep Abrasion (R<<10 nm) Stable - igh P "Micropattern Formation in Supported Lipid Membranes", Jay T. Groves and Steven G. Boxer, Acc. of hem. Res., 35, (2002).

27 hrome Grid 1 mm 5 μm EggP Bilayer 200 μm

28 Different ways to pattern Photolithographic Patterning (Photoactivatable peptides) Microprinting Direct Pipetting Microfluidic Flow "Micropattern Formation in Supported Lipid Membranes", Jay T. Groves and Steven G. Boxer, Acc. of hem. Res., 35, (2002).

29 Microfluidic etworks

30 onstruction Si 2 Si μm SU-8 Mask Mask Si 2 3mm Si 2

31 Evaluation

32 SEM

33 Testing 1 mm Intensity vs Position Intensity Position (mm)

34 onclusions Ellipsometry was used to study the kinetics of VF on Si 2 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

35 Acknowledgments Dr. Alejandro Parra Ellipsometry Dr. aining Zhang PE chemistry ing ao lean-room Processing This work was partially supported by the MRSE Program of the ational Science Foundation under Award o. DMR , the ational Science Foundation IRT Award o. TS , and the Army Research ffice through the Institute for ollaborative Biotechnologies.