Fibronectin / Polyelectrolyte Multilayer Assemblies: Film Formation and Cell Attachment Studies

Corinne Wittmer,* Mark Saltzman,** and Paul Van Tassel* *Dept. of Chemical Engineering, Yale University **Dept. of Biomedical Engineering, Yale University

Biomimetic Coatings
Biomimetic materials possess signaling species mimicking those in vivo Cell membrane protein signaling species cell biomimetic material

Biomimetic coating strategy: signaling species immobilized onto / within coating decouples surface from bulk material properties coating

Layer by Layer (LbL) Method

Create multilayer coating by alternately depositing polycation and polyanion:

1. Polycation 2. Wash

3. Polyanion 4. Wash

Decher (1992)

Incorporation of Biomolecules within Multilayer Film

biomolecule

+ + +

+ + + + + - - - -

- - + + +

- + +

Multilayer films containing biomolecules offer several advantages: Simple to produce Applicable to most biomaterial systems Offer control of biomolecule orientation & conformation Offer temporal control of biomolecule accessibility

Fibronectin (Fn)
A matrix protein inducing cell attachment and spreading Composed of modules, contains cell binding site Biomaterials coated with Fn are excellent candidates as tissue engineering substrates
Matrix Assembly S. Aureus NH2 Fibrin Heparin Cell Binding Site S S

Collagen Gelatin

F1
EDB EDA
RGD

F2

F3

IIICS COOH

Objectives
fibronectin 1. Determine adsorption behavior of Fn on a biocompatible multilayer film extent kinetics reversibility degree of film penetration

2.

Determine cell response to Fnterminated multilayer films cell area cell symmetry

cell

Multilayer Film
Polycation: poly(L-lysine) (PLL) MW = 70,000 - 150,000 pK = 10.5 hydrodynamic diameter 28 - 44 nm (pH 7.4, [NaCl] 100 mM) Polyanion: dextran sulfate (DS) MW 500,000 Buffer: HEPES pH = 7.4 [NaCl] = 100 mM Length Scales: Bjerrum length = .72 nm Debye length = .96 nm
O OSO3OSO3O OSO3-

O NH2-CH- C (CH2)4 NH3+ n

Optical Waveguide Lightmode Spectroscopy (OWLS)

Laser

Measures adsorbed layer dry mass (i.e. mass of polymer only)

Quartz Crystal Microbalance with Dissipation (QCMD)

Adsorbed macromolecules

~
Vibrating sensor crystal Measures adsorbed layer wet mass (i.e. mass of polymer plus mass of solvent)

Multilayer Growth Kinetics: OWLS

600 500

= uffer rinse

1 % DXS1

400
19 % PLL1

= poly(L-lysine) (PLL) = dextran sulfate (DS)

300

1 % D X1

200

% PLL1

100
DXS1

(PLL-DXS)
0 0 PLL1 50 100 150 200 250

Ti e ( in)

Rapid saturation of each layer PLL, DS adsorption steps irreversi le Fil ass ~ exp [ # of layers ]

Multilayer Growth Kinetics: OWLS vs. QCMD

Dry film mass via OWLS (Si iO2 substrate)
600

Wet film mass via QCMD (SiO2 substrate)

7000

170% D S1

Asorbed Density (ng/cm )

400
190% PLL1

300
140% DE 1

200
130% PLL1

(PLL-D S)3
D S1

0 0 PLL1 50 100 150 200 250

ime (min)

Large wet mass increase during DS steps Wet mass decrease during PLL steps > 90% film mass is water !

100

500

= buffer rinse

6000 5000 4000 3000 2000 1000 0 0 50 100 150 200

ime (min)

/
1000 900 800 700 600 500 400

il

1000

Buffer rinse
ka = 6x10-5 cm/s

900 800

00 600 500 00 300 00 0 100

300 200 100 0 0 50 100 150 200

250

300

50

100

150

00

50

ti

( i )

i e

in

rpti

xhi it

initial rate, initial extent, reversibility

on PLL versus DS terminated film Why? DS layer very hydrated, thus resistant to protein adsorption

- X ) n 3

s r e

ensi

ka = 1x10-4 cm/s

n
300