Immobilized heparin via a long chain poly(ethylene oxide) spacer for protein and platelet compatibility

Poly(ethylene oxide) has some unique solubility and hydrogen-bonding characteristics which have been used to explain its apparent inertness with regard to blood protein absorption and platelet interactions. A method has been developed to immobilize long chains of the hydrophilic and amorphous polymer by derivatization of the end hydroxyl groups allowing attachment to and extension out from the surface. This creates an "excluded volume" which is inaccessible to large protein molecules and cells. Four derivatives of poly(ethylene oxides) (PEO) have been synthesized: PEO-bis isocyanate, PEO-bis isothiocyananate, PEO-bis chloroformate, and PEO bis thiochloroformate. The cyanate derivatives were synthesized using the lithium salt of cysteamine to form diamine-terminated PEO chains which were subsequently reacted with phosgene of thiophosgene. The chloroformate derivatives were produced by directly reacting the hydroxyl groups of the PEO with phosgene and thiophosgene. Molecular weights of PEO ranging from 1,000 to 18.000 daltons were used. Heparin was immobilized onto the reactive free ends of these PEO derivatives via the free amine and hydroxyl groups of the heparin molecule, permitting extension of the anticoagulant from the surface, allowing it to assume its native and active conformation of interaction with specific blood proteins. Surfaces prepared by the method were characterized by x-ray photoelectron spectroscopy and found to be stable in aquenos environments. Minimal nonspecific protein absorption and adhesion of platelets to the PEO surfaces without heparin was demonstrated by Total Internal Reflection Fluorescenne Spectroscopy (TIRF) and 125-iodine labeled proteins. It was found that there is a small inverse relationship between chain length of the PEO and the quantity of protein absorbed onto the surface, confirming earlier reports. Platelet retention has been studied by exposing platelet rich plasma to derivatized glass beads and has demonstrated significantly less retention than the control surfaces. Quantitation was accomplished using tritiated heparin. The activity of the heparin was shown by the ability of these surfaces to bind antithrombin-III and by significantly increased whole blood clotting times and activated partial thromboplastin times. The results reported her suggest that long chain poly(ethylene oxide) offers a relatively inert surface for the immobilization of biologically active molecules, minimizing nonspecific interactions and providing a mechanism for these active molecules to react freely, mimicking their solution activities.