RESUMEN
The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, "sequential" and "direct" were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG-CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG-CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the "target" protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time.
Asunto(s)
Anticoagulantes/química , Proteínas de Plantas/química , Polietilenglicoles/química , Proteínas/químicaRESUMEN
In previous work using gold as a model substrate, we showed that modification of surfaces with poly(ethylene glycol) (PEG) and corn trypsin inhibitor (CTI) rendered them protein resistant and inhibitory against activated factor XII. Sequential attachment of PEG followed by CTI gave superior performance compared to direct attachment of a preformed PEG-CTI conjugate. In the present work, a sequential method was used to attach PEG and CTI to a polyurethane (PU) substrate to develop a material with applicability for blood-contacting medical devices. Controls included surfaces modified only with PEG and only with CTI. Surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy. The surface density of CTI was in the range of a monolayer and was higher on the PU substrate than on gold reported previously. Biointeractions were investigated by measuring fibrinogen adsorption from buffer and plasma, factor XIIa inhibition and plasma clotting time. Both the PU-PEG surfaces and the PU-PEG-CTI surfaces showed low fibrinogen adsorption from buffer and plasma, indicating that PEG retained its protein resistance when conjugated to CTI. Although the CTI density was lower on PU-PEG-CTI than on PU modified only with CTI, PU-PEG-CTI exhibited greater factor XIIa inhibition and a longer plasma clotting time, suggesting that PEG facilitates the interaction of CTI with factor XIIa. Thus sequential attachment of PEG and CTI may be a useful approach to improve the thromboresistance of PU surfaces.
Asunto(s)
Proteínas Sanguíneas/administración & dosificación , Materiales Biocompatibles Revestidos/administración & dosificación , Proteínas de Plantas/administración & dosificación , Polietilenglicoles/administración & dosificación , Poliuretanos/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Proteínas Sanguíneas/química , Materiales Biocompatibles Revestidos/síntesis química , Materiales Biocompatibles Revestidos/química , Factor XIIa/química , Factor XIIa/metabolismo , Fibrinógeno/química , Fibrinógeno/metabolismo , Humanos , Espectroscopía de Fotoelectrones , Proteínas de Plantas/química , Polietilenglicoles/química , Poliuretanos/química , Propiedades de SuperficieRESUMEN
Blood contacting surfaces bind plasma proteins and trigger coagulation by activating factor XII (FXII). The objective of this work was to develop blood contacting surfaces having the dual properties of protein resistance and inhibition of coagulation. Gold was used as a model substrate because it is amenable to facile modification using gold-thiol chemistry and to detailed surface characterization. The gold was modified with both polyethylene glycol (PEG) and corn trypsin inhibitor (CTI), a potent and specific inhibitor of activated FXII (FXIIa). Two methods of surface modification were developed; sequential and direct. In the sequential method PEG was first chemisorbed on gold; CTI was then attached to the PEG. In the direct method a conjugate of PEG and CTI was first prepared; the conjugate was then immobilized on gold. The surfaces were characterized by water contact angle and XPS. Biointeractions with the modified surfaces were assessed by measuring fibrinogen adsorption from buffer and plasma and by immunoblot analysis of eluted proteins after plasma exposure. Inhibition of FXIIa, autoactivation of FXII, and clotting times of plasma in contact with the surfaces were also measured. Both the sequential and direct surfaces showed reduced protein adsorption, increased FXIIa inhibition and longer clotting times compared with controls. Although the CTI density was lower on surfaces prepared using the sequential method, surfaces so prepared exhibited greater CTI activity than those generated by the direct method. It is concluded that the activity of immobilized PEG-CTI depends on the method of attachment and that immobilized CTI may be useful in rendering biomaterials more blood compatible.
Asunto(s)
Proteínas de Plantas/química , Polietilenglicoles/química , Propiedades de Superficie , Zea mays/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this work, we hypothesize that a surface modified with both polyethylene glycol (PEG) and hirudin may provide a non-fouling, thrombin-neutralizing surface suitable for blood contacting applications. With gold as a model substrate we used two different approaches to the preparation of such a surface: (1) a "direct" method in which PEG was conjugated to hirudin and the conjugate was then immobilized on the gold; (2) a "sequential" method in which PEG was immobilized on the gold and hirudin then attached to the immobilized PEG. The surfaces were characterized by water contact angle, ellipsometry and XPS. The biological properties were investigated by measuring protein adsorption (fibrinogen and thrombin) from buffer and plasma; thrombin inhibition was measured using a chromogenic substrate assay. Hirudin immobilization was found to be more efficient on surfaces prepared by the "direct" method. "Sequential" surfaces, however, despite having a lower density of hirudin, showed greater biological activity (thrombin binding and inhibition).
Asunto(s)
Fibrinógeno/metabolismo , Hirudinas/química , Hirudinas/farmacología , Trombina/antagonistas & inhibidores , Trombina/metabolismo , Adsorción/efectos de los fármacos , Oro/química , Humanos , Propiedades de SuperficieRESUMEN
Protein adsorption and platelet adhesion properties of polyurethane biomaterials are important considerations for blood-contacting applications. Although the presence of ionic groups on the surface of biomaterials is believed to influence their blood response, their exact role is not known. The objective of this work was to study the protein adsorption and platelet adhesion properties of ion-containing polyurethane biomaterials. Thus, we prepared polyurethanes that contained ions either on the soft segment or hard segment and investigated their in vitro protein adsorption and platelet adhesion. The presence of ions increased the amount of adsorbed proteins and adhered platelets on the synthesized polyurethanes. Whereas albumin and lysozyme adsorption were independent of the location of the ions (soft vs. hard segments), fibrinogen adsorption was strongly dependent on the location of the ions. Platelet adhesion, on the other hand, was found to be less dependent on the location of the ions within the polyurethane structure. This is the first evidence to unequivocally demonstrate the exact role of ions on protein adsorption and platelet adhesion. Taken together, our study suggests that in the absence of known biocompatible chains such as polyethyleneoxide, ion-containing polyurethanes do not demonstrate improved blood compatibility. Therefore, we conclude that ion incorporation into polyurethanes may not be a viable approach to design polyurethane biomaterials for blood-contacting applications.