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<p>Surface-induced thrombosis is a continuing issue in the development of biomaterials for blood contacting applications. Protein adsorption is a key factor in thrombosis since it occurs rapidly upon contact of a material with blood, initiating coagulation and other adverse reactions including platelet adhesion. The research presented in this thesis explores the use of a unique antithrombin-heparin covalent complex (ATH) for surface modification to provide antithrombogenicity. ATH was tethered to surfaces by various methods. Polyethylene oxide (PEO) was investigated as a linker-spacer molecule for surface attachment of ATH as well as for its antifouling properties.</p> <p>In the first phase of the work gold was used as a model substrate. ATH was attached by three different methods: direct attachment, attachment via a short chain linker, and attachment via PEO. Analogous heparin-modified surfaces were prepared for comparison. Surfaces were characterized using contact angle measurements, x-ray photoelectron spectroscopy (XPS), ellipsometry and quartz-crystal microbalance (QCM). The data suggested that the heparin moiety of ATH was directed away from the surface, in an orientation allowing ready interaction with blood components. The ATH-modified surfaces showed greater antithrombin binding than the heparin-modified surfaces as measured by radioactive labelling and Western blotting analysis. Antithrombin binding was found to occur predominantly through the active pentasaccharide sequence of the heparin moiety of ATH, demonstrating the potential of the ATH for catalytic anticoagulant function. From measurements of the ratio of total heparin to active heparin (anti-factor Xa assay), ATH-modified surfaces were shown to have greater bioactivity than heparin-modified surfaces. The adhesion of platelets to gold and modified gold surfaces was measured from flowing whole blood <em>in vitro</em> using a cone-and-plate device and was lower on all of the modified surfaces compared to bare gold. PEO-ATH surfaces were also shown to prolong plasma clotting times compared to control and heparinized surfaces.</p> <p>In subsequent work, surface modification methods were developed for polyurethane (PU) substrates. Isocyanate groups were introduced into the PU surface for attachment of PEO and ATH was attached to the “distal” end of the PEO. Surfaces using PEO of varying molecular weight and end group were investigated to determine conditions for maximum anticoagulant activity and minimum non-specific protein adsorption. Surfaces were characterized using contact angle measurements and XPS, and protein interactions were studied using radiolabelling. The optimum balance of bioactivity and protein resistance was found to occur with PEO of low to mid range MW (ie. MW 300-600). These PU-PEO-ATH surfaces showed low fibrinogen adsorption and high selectivity for antithrombin. Consistent with results using gold substrates, platelet adhesion remained low when ATH was attached to polyurethane surfaces grafted with PEO. A hetero-bifunctional amino-carboxy-PEO (PEO-COOH surface) was compared with a “conventional” homo-bifunctional dihydroxy-PEO (PEO-OH surface) with respect to their effectiveness as linkers for attachment of ATH. The PEO-COOH-ATH surface was shown to bind slightly greater amounts of antithrombin, indicating higher catalytic anticoagulant activity. Thrombin binding was measured to determine whether the surfaces could provide direct anticoagulant activity. The PEO-OH-ATH surface bound high amounts of thrombin, indicating potential for direct thrombin inhibition. It is hypothesized that the PEO properties (MW and functional end group) may have an effect on the orientation of ATH on the surface thus influencing its "preference" for catalytic vs. direct anticoagulant function.</p> <p>This thesis provides new information regarding the interactions of proteins and platelets with ATH immobilized on biomaterials. ATH-modified surfaces were superior to analogous heparin-modified surfaces with respect to antithrombin binding and catalytic anticoagulant ability. Immobilized ATH was also shown to bind thrombin, suggesting potential for direct anticoagulant activity. It can thus be seen as a unique surface modifier with dual functioning anticoagulant activity. The modification of polyurethane with ATH using PEO as a protein resistant linker-spacer, may provide a material of improved antithrombogenicity for the construction of blood contacting devices.</p> / Doctor of Philosophy (PhD)

Biofouling is an ongoing problem in the development and usage of biomaterials for biomedical implants, microfluidic devices, and water-based sensors. Antifouling coatings involving surface modification of biomaterials is widely utilized to reduce unwanted protein adsorption and cell adhesion. Surface modification strategies, however, are reliant on the working material’s chemical properties. Thus, published procedures are often not applicable to a wide range of material classes. This constitutes a serious limitation in using surface modification on assembled multi-material devices, i.e on whole device modification. The objective of this research is to develop an antifouling coating with non-aggressive reaction conditions that can universally modify polymers and other material classes. Two strategies using polydopamine (PDA) as an anchor for polyethylene glycol (PEG) surface attachment were investigated: (1) PDA-PEG backfilled with bovine serum albumin (BSA), and (2) PDA-PEG with light activated perfluorophenyl azide (PFPA) conjugated to the PEG. Three materials varying in surface wettability were studied to evaluate the coatings for multi-material applications: porous polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass cover slips. Atomic force microscopy (AFM) and ellipsometry studies revealed substantial structural differences of PDA. Differences in PDA surface roughness affected PEG grafting in solution (the first method), with higher PEG coverage achieved on PC with intermediate surface roughness to PDMS and glass. Radiolabeled Fg adsorption and E. coli adhesion experiments showed reduced fouling on all PDA-PEG modified materials when backfilled with BSA. The ability for BSA to penetrate the PEG layer indicated that low PEG grafting densities were achieved using this grafting-to approach. The use of a photoactive labeling agent, PFPA, to tether PEG was proposed to improve PEG grafting on PDA. The PFPA-PEG modification protocol was optimized by quantifying Fg adsorption. Two treatments of PFPA-PEG were required to fully block PDA active sites. Fg adsorption was not significantly improved on PFPA-PEG modified PC and glass when backfilled with BSA, indicating sufficient PEG coverage of PDA. High Fg adsorption on PFPA-PEG surfaces indicate that high density PEG brushes were still not achieved with this method. PDMS surfaces were damaged with this procedure due to increased surface handling in the protocol. This is the first, to our knowledge, successful demonstration of PFPA modification on PDA surfaces. Photopatterning of polymer-based materials can be achieved, providing opportunities for utilising new materials in cell patterned platforms. Due to low PEG coverage on PDA surfaces from solution and using PFPA, ultra-low protein adsorption cannot be achieved using these aqueous-based methods. Antifouling modifications using PDA and PEG should be applied for short-term cell studies. / Thesis / Master of Applied Science (MASc)