Investigating the Origins of Protein-Surface

Adsorption:Experimental Results

Ellipsometry: A Macroscopic Measure of Protein Surface Adhesion

i

t

rRparallel / RperpendicularEi =

Ellipsometery measures theRatio of the electric fields ofThe reflectid waves parallel and perpendicular to theInterface; from this you mayExtrapolate the thickness of The interface

Alternatively: ellipsometry Measures the abruptness ofChange in refractive index From the surrounding medium (air, e.g.) to the substrate, and from this extrapolates the film thickness

Force Microscopy: A Microscopic Measure of

Protein Adhesion • Protein is

covalently attached to the probe tip

• Adhesion is measured on various substrates/SAMs of different degrees of hydrophilicity

Protein vs Substrate

• Three different blood plasma proteins studied: Albumin (Alb), Immunoglobulin G (IgG), and Fibrinogen (Fib)

• Four different SAMs studied; in order of increasing hydrophilicity: -CH3, -OH, -NH2, -COOH

• Protein-protein, protein-SAM, and SAM-SAM interactions compared

Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.

The experiment:

SAM-SAM & Protein-Protein Interactions

Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.

Protein-SAM Interactions

Schematic of protein adhesion

Experimental results

Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.

Not All Proteins Were Created Equal

• In order of increasing SAM affinity for each protein:

– Alb, IgG: -CH3 >> (-OH, -NH2) > -COOH

– Fib:-CH3 >> -OH > -NH2 > -COOH

– Fib > Alb, IgG on all surfaces except -COOH

Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.

The Importance of Conformation

* The extent of protein interaction depends not only on the type of SAM, but also on the SAM conformation

Kidoaki, S.; Nakayama, Y.; Matsuda, T. Langmuir 2001, 17, 1080-1087.

Re: Methods for Counteracting Protein-

Surface Interaction with Polymer Coatings

• Dense polymer coatings (low )

• Long polymer chains (large N)

d

R N

Uout may be manipulated by varying N or Uin is primarily controlled by varying

Effect of and L on Surface Interaction

Forces• The polymer chains in a brush

are not fully extended:

• There is a point at which the polymer layer becomes incompressible: Do

where D’ = Dexperimental and

PMMA)for (0.6 1contour

mequilibriu

L

L

PMMA)MW high for (0.8 1eqL

D

Yamaoto, Shinpei; Muhammad, Ejaz; Yoshinobu, Tsujii; Matsumoto, Mutsuo; Fukuda, Takeshi. Macromolecules 2000, 33, 5602-5607.Yamaoto, Shinpei; Muhammad, Ejaz; Yoshinobu, Tsujii; Fukuda, Takeshi. Macromolecules 2000, 33, 5608-5612.

oD D'D

0.5)-0.3(n n

The Effect of and L on Protein Adhesion to PEO

• At very high surface densities , SAMs will resist adsorption of all types of proteins, with universal resistance achieved at lower for higher molecular weight (larger L) SAMs

• L is not as influential as • The highest L at optimum is most

effective at protein resistance• Adhesion is temperature-dependent

Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid and Interface Sci., 142 (1), 149-158 (March1, 1991).Jeon, S. I.; Andrade, J. D. J. Colloid and Interface Sci., 142 (1), 159-166 (March 1, 1991).Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc., 1993, 115 10714-10721.

The Effect of the Substrate on the SAM

Conformation• PEO on gold in

aqueous solution is predominantly in a helical conformation stabilized by H-bonding

• On silver, however, the binding sites are so close that the helix is sterically hindered

Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.

Fibrinogen Adhesion

Mica

C16H33-Au

EG3-Au

EG3-Ag

Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.

Tip-Surface Electrostatics: the Effect of Ions in

SolutionSi3N4 tip + EG3 C16 tip + EG3

DI H2O PBS

Au

Ag

Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.

Tip-Surface Electrostatics: the Effects of Ionic

Strength and Molecular Weight

Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. A. Chem. Soc. 1999, 121, 10134-10141.Feldman, K.; Haehner, G.; Spencer, N. D.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141.

Polymer Architectures

Linear Comb

Star

Effect of Chain Architecture on Protein

Adsorption

Mayes, A. M.; Irvine, D. J.; Griffith, L. G. Mat. Res. Soc. Symp. Proc. 1998, 530, 73-84.

In contrast to linear polymers, the center mass for star polymers lies at some distance away from the surface.

This results in a much more energetically favored state for protein adhesion at the surface, once diffusion through the polymer layer is achieved

Measuring Protein Adhesion with the Surface

Force Apparatus

Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).

Compression Leads to Protein-Surface Binding

• A: Protein resistance still observed at low compressive loads ( <4kT )

• B: Under sufficient compressive loads ( >4kT ) attractive interactions dominate

Note: Derjaguin approximation: kT

R

F2

2

per chain

Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).

Conclusions• Design of biomaterials is challenged by the

complicated, interrelated factors involved in of achieving biocompatibility: i.e. protein resistance vs cell specificity

• Because they are easily tailored to meet specific chemical needs, polymers are often used as coatings on compliance-matched devices

• Optimization of polymer coatings is a delicate balance among a) size, architecture, and even supramolecular structure of the polymer, b) the density of the polymer layer, c) the type of underlying substrate and its electrostatic properties, d) the identity of the targeted proteins, and e) the magnitudes of the forces the biomaterial will be subjected to in vivo