Investigating the Origins of Protein-Surface Adsorption:

1. Investigating the Origins of Protein-Surface Adsorption: Experimental Results

2. Ellipsometry: A Macroscopic Measure of Protein Surface Adhesion Ellipsometery measures the Ratio of the electric fields of The reflectid waves parallel and perpendicular to the Interface; from this you may Extrapolate the thickness of The interface Alternatively: ellipsometry Measures the abruptness of Change in refractive index From the surrounding medium (air, e.g.) to the substrate, and from this extrapolates the film thickness i r Rparallel Ei = / Rperpendicular t

3. 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

4. Protein vs Substrate The experiment: • 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. Langmuir1999, 15, 7639-7646.

5. SAM-SAM & Protein-Protein Interactions Kidoaki, S.; Matsuda, T. Langmuir1999, 15, 7639-7646.

6. Protein-SAM Interactions Experimental results Schematic of protein adhesion Kidoaki, S.; Matsuda, T. Langmuir1999, 15, 7639-7646.

7. 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. Langmuir1999, 15, 7639-7646.

8. 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. Langmuir2001, 17, 1080-1087.

9. Re: Methods for Counteracting Protein-Surface Interaction with Polymer Coatings • Dense polymer coatings (low s) • Long polymer chains (large N) Uoutmay be manipulated by varying N or s Uinis primarily controlled by varyings R a N da s

10. Effect of s 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 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.

11. The Effect of s and L on Protein Adhesion to PEO • At very high surface densities s, SAMs will resist adsorption of all types of proteins, with universal resistance achieved at lower s for higher molecular weight (larger L) SAMs • L is not as influential as s • The highest L at optimum s 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.

12. 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.

13. Fibrinogen Adhesion Mica EG3-Au C16H33-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.

14. Tip-Surface Electrostatics: the Effect of Ions in Solution C16 tip + EG3 Si3N4 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.

15. 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.

16. Polymer Architectures Linear Comb Star

17. Effect of Chain Architecture on Protein Adsorption 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 Mayes, A. M.; Irvine, D. J.; Griffith, L. G. Mat. Res. Soc. Symp. Proc. 1998, 530, 73-84.

18. Measuring Protein Adhesion with the Surface Force Apparatus Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. USA, 94, 8399-8404 (August 1997).

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

20. 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