Fibronectin Polyelectrolyte Multilayer Assemblies:

1
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
2
Biomimetic Coatings
Biomimetic materials possess signaling species
mimicking those in vivo
Cell membrane protein
signaling species
biomimetic material

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

coating
3
Layer by Layer (LbL) Method
Create multilayer coating by alternately
depositing polycation and polyanion
Decher (1992)
4
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

5
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

6
Objectives
fibronectin

• Determine adsorption behavior of Fn on a
  biocompatible multilayer film
• extent
• kinetics
• reversibility
• degree of film penetration
• Determine cell response to Fn-terminated
  multilayer films
• cell area
• cell symmetry

7
Multilayer Film
O

• 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

NH2-CH- C
(CH2)4
NH3
n
8
Optical Waveguide Lightmode Spectroscopy (OWLS)
Measures adsorbed layer dry mass (i.e. mass of
polymer only)
9
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)
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Multilayer Growth Kinetics OWLS
poly(L-lysine) (PLL) dextran sulfate
(DS)

• Rapid saturation of each layer
• PLL, DS adsorption steps irreversible
• Film mass exp of layers

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Multilayer Growth Kinetics OWLS vs. QCMD
Dry film mass via OWLS (SiTiO2 substrate)
Wet film mass via QCMD (SiO2 substrate)

• Large wet mass increase during DS steps
• Wet mass decrease during PLL steps
• gt 90 film mass is water !

12
Fn on PLL / DS Film
Buffer rinse
ka 6x10-5 cm/s
(PLL-DS)3-Fn
(PLL-DS)2-PLL-Fn
Fn adsorption exhibits ? initial rate, ?
initial extent, ? reversibility on PLL versus
DS terminated film Why? DS layer very hydrated,
thus resistant to protein adsorption