AVS 49th International Symposium
    Biomaterials Tuesday Sessions
       Session BI-TuP

Paper BI-TuP6
Derivatization and Surface Characterization of poly(2-hydroxyethyl methacrylate) for Oriented Protein Immobilization

Tuesday, November 5, 2002, 5:30 pm, Room Exhibit Hall B2

Session: Biointerfaces and Surfaces I
Presenter: J.L. Schwartz, University of Washington
Authors: J.L. Schwartz, University of Washington
S.M. Martin, University of Washington
D.G. Castner, University of Washington
C.M. Giachelli, University of Washington
B.D. Ratner, University of Washington
Correspondent: Click to Email
When biomaterials are placed in the body they are immediately covered with a random layer of mixed proteins. This may trigger the foreign body reaction leading to collagenous tissue encapsulating the biomaterial. To circumvent this, many schemes have been developed to pre-immobilize a protein or peptide onto the surface intended to subdue the inflammatory response. The goal of this study is to immobilize osteopontin (OPN) in a single orientation such that the active site is presented to the surface for optimal cellular interaction. Proteins can be immobilized via a primary amine from a lysine unit, but due to the number and distribution of lysine in OPN there is no control over the final orientation. A different approach is to immobilize OPN engineered with a polyhistidine tail, which can interact with the coordination sites of a divalent metal ion. The recombinant six-histidine (His-tag) sequence does not hinder protein activity and can be placed at a characteristic site on the protein of interest. The divalent metal ion, Ni@super 2+@, binds tightly to nitrilotriacetic acid (NTA), a metal chelating agent covalently attached to poly(2-hydroxyethyl methacrylate) (pHEMA) via a N,N-carbonyldiimidazole (CDI) intermediate reaction step. The choice of pHEMA as the immobilizing substrate was due to the abundance of hydroxyl groups on the surface as well as low non-specific protein adsorption. These surfaces were characterized by XPS and ToF-SIMS before and after chemical derivatization as well as after each reaction step. Derivatization with fluorine containing molecules was used to probe hydroxyl, carboxyl, and imidazole carbamate availability with XPS. The amount of protein immobilized to pHEMA was quantified by @super 125@I radiolabeled OPN and the protein was tested for retained biological activity.