To improve blood compatibility of biomaterials, we have immobilized bioactive agents, such as heparin, onto polymer surfaces.' Heparin immobilization was achieved by grafting heparin and spacer groups onto soluble polymer chains which can then be coated onto existing surfaces.2 Another method is to directly couple heparin and spacer groups onto solid polymer surfaces as in situ m~dification.~ Both systems have proven effective in decreasing fibrin formation, as demonstrated in in vitro, ex vivo, and in vivo evaluation^.^ Although success has been made in this area, one of the limitations is the restricted coupling for heparin immobilization. Immobilization should be optimized to increase heparin concentration on the biomaterial surface and preserve both the native chemical composition (amounts of carboxylic acid and sulfate groups) and conformation of immobilized heparin to maintain biological activity.' A possible way to increase the amount of immobilized heparin is to amplify the reactive sites on the polymer surface through which heparin can be coupled.
Larm et a1.6 modified polyethylene tubing with branched high-molecularweight polyethyleneimine (PEI) and heparin in order to increase the surface concentration for anticoagulating activity. A surface coating containing PEI coupled with heparin was applied to p~lyurethane.~ Both studies demonstrated stable heparin attachment and maintained the bioactivity of heparin. PEI has been shown to be a suitable amplifying agent due to its abundance of amine groups for derivatization by -NCO.
We attempted a novel method to increase the concentration of immobilized heparin on the surface of polyurethane (PU). A polyfunctional compound was first grafted on PU surface through diisocyanate groups. Polyethylene oxide (PEO) spacer was then coupled to the grafted surface followed by heparin immobilization as shown in Figure .
Hexamethylene diisocyanate (HMDI, Sigma Chemical Co.), tolylene 2,4 diisocyanate (TDI, Aldrich Chemical Co.), methylenediphenylene diisocyanate (MDI, Polysciences, Inc.), methyl sulfoxide (anhydrous, Aldrich Chemical Co.), di-n-butyl tin dilaurate (DBTD, Polysciences, Inc.), sodium heparin (179.1 IU/mg, Diosynth Inc.), and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma Chemical Co.) were used as supplies. D- sorbitol (SBL, Aldrich Chemical Co.), pentaethylenehexamine (PEHA, Aldrich Chemical Co.), poly(viny1 alcohol) (PVA, 88% hydrolyzed, M.W., 77,000-79,000, Aldrich Chemical Co.), and PEI (30% water solution, M.W, 50,000-