AbstractSurface modification with bioactive agents capable of combating thrombosis is a widely used strategy for developing antithrombotic biomaterials. However, exposure of the blood to the antithrombotic agent on the material surface may cause hemostatic disorders under normal conditions. Ideally an implanted biomaterial should respond appropriately on demand to a specific change in the physiologic environment, as happens in the body itself. In the present study, a thrombosis-responsive surface coating with the ability to lyse fibrin as it forms is reported. The coating consists of nanocapsules (NCs) in which the fibrinolysis activator t-PA is encapsulated in a thrombin-degradable hydrogel shell. The t-PA NCs are attached to several materials covalently through a polydopamine adhesive layer. The resulting surfaces are treated with the antifouling agent glutathione (GSH) to prevent further interactions with blood/plasma components. The t-PA NCs/GSH-coated surface is stable and remain inert in normal plasma environment while releasing t-PA and promoting fibrinolysis when thrombin is present. The fibrinolytic activity increases with increasing thrombin concentration, and therefore presumably with the extent of thrombosis. This work constitutes the first report of an antithrombotic coating whose function is triggered and regulated, respectively, by the appearance of thrombin and the extent of coagulation.

A major issue in the therapeutic use of tissue plasminogen activator (t-PA) for the treatment of thrombotic diseases is its very short half-life in the circulation due to the effects of inhibitors. The present study aims to resolve the issue using a t-PA/gold nanoparticle (t-PA/AuNP) conjugate prepared via bio-affinity ligation under physiological conditions. The ligation is based on the specific interactions between t-PA and ε-lysine (a ligand that has affinity to a specific domain in t-PA) immobilized on the AuNP surface through polyvinyl pyrrolidone (PVP) as a spacer. The conjugate can not only retain almost full enzymatic activity and clot dissolving efficiency, but also protect t-PA from inhibition by PAI-1 to some extent as compared with free t-PA in vitro. Moreover, the conjugate showed prolonged circulation time in vivo.

Surface modification with affinity ligands capable of capturing bioactive molecules in situ is a widely used strategy for developing biofunctional materials. However, many bioactive molecules, for example zymogens, exist naturally in a “quiescent” state, and become active only when “triggered” by specific activators. In the present study, in situ activation of a surface-integrated zymogen was achieved by introducing affinity ligands for both the zymogen and its activator. Specifically a dual affinity surface was designed for the integration of plasminogen (Plg) and tissue plasminogen activator (t-PA). This surface was expected to have plasmin-generating and, therefore, fibrinolytic properties. A polyurethane surface was modified with a copolymer of 2-hydroxyethyl methacrylate and 1-adamantan-1-ylmethyl methacrylate poly(HEMA-co-AdaMA). The affinity ligands, ARMAPE peptide (for t-PA) and ε-lysine-containing β-cyclodextrin (β-CD-(Lys)7) (for Plg), were attached in sequence via covalent bonding and host–guest interactions, respectively. The resulting surfaces were shown to have high binding capacities for both t-PA and Plg while resisting nonspecific protein adsorption. Pre-loading with t-PA followed by Plg uptake from plasma generated plasmin and thus endowed the surface with fibrinolytic activity. In general the incorporation of dual affinity ligands to achieve surface-promoted bioactivity is a promising approach for the development of biofunctional materials. The method reported herein for the sequential attachment of plasminogen and t-PA affinity ligands can be extended to systems of multiple ligands generally.

The concept of enzyme immobilization via an inhibitor-derived peptide was developed. This method of immobilization was shown to be advantageous over physical adsorption and covalent bonding in retaining the enzymatic activity. Moreover, the surface-immobilized enzyme exhibited resistance against its inhibitor due to the occupation of an inhibitor binding site on the enzyme.

A hexapeptide derived from an enzyme inhibitor was used as an affinity ligand for the conjugation of a hydrophilic polymer to the enzyme. The peptide targeted the polymer to the “berth” of the inhibitor in the enzyme, affording the enzyme resistance to the inhibitor without affecting the enzymatic activity.

The extent of protein adsorption is an important consideration in the biocompatibility of biomaterials. Various experimental methods can be used to determine the quantity of protein adsorbed, but the results usually differ. In the present work, self-assembled monolayers (SAMs) were used to prepare a series of model gold surfaces varying systematically in water wettability, from hydrophilic to hydrophobic. Three commonly used methods, namely, surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), and 125I-radiolabeling, were employed to quantify fibrinogen (Fg) adsorption on these surfaces. This approach allows a direct comparison of the mass of Fg adsorbed using these three techniques. The results from all three methods showed that protein adsorption increases with increasing surface hydrophobicity. The increase in the mass of Fg adsorbed with increasing surface hydrophobicity in the SPR data was parallel to that from 125I-radiolabeling, but the absolute values were different and there does not seem to be a “universally congruent” relationship between the two methods for surfaces with varying wettability. For QCM-D, the variation in protein adsorption with wettability was different from that for SPR and radiolabeling. On the more hydrophobic surfaces, QCM-D gave an adsorbed mass much higher than from the two other methods, possibly because QCM-D measures both the adsorbed Fg and its associated water. However, on the more hydrophilic surfaces, the adsorbed mass from QCM-D was slightly greater than that from SPR, and both were smaller than from 125I-radiolabeling; this was true no matter whether the Sauerbrey equation or the Voigt model was used to convert QCM-D data to adsorbed mass.

It is well-known that extracellular matrix (ECM) proteins mediate cell/surface interactions. However, introduction of a specific surface topography may disturb the correlation between ECM proteins adsorption and cells adhesion on a given surface. In present study, lotus-leaf-like topography was introduced on the surface of a biodegradable material, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Protein adsorption and cell interactions with this lotus-leaf-like surface (designated PHBHHx-L) were investigated. Water contact angle data indicated that the hydrophobicity of PHBHHx was enhanced by the introduction of lotus-leaf-like topography. The adsorption of extracellular matrix proteins (fibronectin and vitronectin) on PHBHHx-L was measured by enzyme linked immunosorbent assay (ELISA). Compared with flat PHBHHx, adsorption on the PHBHHx-L surface increased by ∼260% for fibronectin and ∼40% for vitronectin. In contrast, fibroblast and endothelial cell adhesion and proliferation were reduced on the PHBHHx-L compared to the flat polymer surface. These results suggest that the inhibition of cell adhesion and proliferation caused by the lotus-leaf-like topography dominates over the effect of the adsorbed adhesive proteins in promoting adhesion and proliferation. It can be concluded that the lotus-leaf-like topography plays a dominant role in cell/PHBHHx-L interactions. The present findings indicate the complexity of the interplay among surface topography, adsorbed proteins, and cell–surface interactions.Keywords: cell proliferation; lotus-leaf-like; poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); protein adsorption; surface topography;

A new strategy to regulate the attachment of plasminogen and its activator, t-PA, to a polyurethane surface is reported. The method is based on the one-step graft copolymerization of a lysine-containing methacrylic monomer and hydroxyethyl methacrylate (HEMA). The copolymer-modified PU surfaces showed high capacity for plasminogen and t-PA binding. The release of pre-adsorbed t-PA from the lysine-containing PU surfaces in contact with plasma was also investigated. It was shown that the interactions of plasminogen and t-PA on this type of surface can be controlled simply by changing the monomer feed ratio, and thus the relative quantities of HEMA and lysine in the grafts. This approach may be useful for the development of fibrinolytic, blood-contacting materials and, more generally, for controlling protein-surface interactions for biocompatibility.

The control of protein/surface interactions by external stimuli is often required in bioapplications such as bioseparation and biosensors. Although regulation of protein adsorption has been achieved on the surfaces modified with stimuli-responsive polymers, controlled protein adsorption is still challenging for a target protein in a multiprotein system. The present study developed a concept of surface design for the controlled adsorption of a specific protein from plasma by combining a thermoresponsive polymer with an affinity ligand on the surface. In this regard, a polyurethane (PU) surface was modified with the copolymer of N-isopropylacrylamide (NIPAAm) and a ε-lysine-containing monomer (LysMA). ε-Lysine is a specific ligand for plasminogen that was used as the model “target protein” in this study. The PU-P(NIPAAm-co-Lys) surfaces exhibited distinct thermoresponsivity of plasminogen adsorption from plasma with a larger quantity adsorbed at 37 °C than at 23 °C. By contrast, the surfaces showed a low level of adsorption for other plasma proteins at both temperatures. In addition, plasminogen adsorbed on a PU-P(NIPAAm-co-Lys) surface could be partly desorbed by lowering the temperature, and the activity of plasminogen adsorbed was well preserved. We believe that the concept developed in this study can be extended to other proteins by combining PNIPAAm and specific ligands with affinities for the proteins of interest.

The concept of enzyme immobilization via an inhibitor-derived peptide was developed. This method of immobilization was shown to be advantageous over physical adsorption and covalent bonding in retaining the enzymatic activity. Moreover, the surface-immobilized enzyme exhibited resistance against its inhibitor due to the occupation of an inhibitor binding site on the enzyme.

A hexapeptide derived from an enzyme inhibitor was used as an affinity ligand for the conjugation of a hydrophilic polymer to the enzyme. The peptide targeted the polymer to the “berth” of the inhibitor in the enzyme, affording the enzyme resistance to the inhibitor without affecting the enzymatic activity.

A major issue in the therapeutic use of tissue plasminogen activator (t-PA) for the treatment of thrombotic diseases is its very short half-life in the circulation due to the effects of inhibitors. The present study aims to resolve the issue using a t-PA/gold nanoparticle (t-PA/AuNP) conjugate prepared via bio-affinity ligation under physiological conditions. The ligation is based on the specific interactions between t-PA and ε-lysine (a ligand that has affinity to a specific domain in t-PA) immobilized on the AuNP surface through polyvinyl pyrrolidone (PVP) as a spacer. The conjugate can not only retain almost full enzymatic activity and clot dissolving efficiency, but also protect t-PA from inhibition by PAI-1 to some extent as compared with free t-PA in vitro. Moreover, the conjugate showed prolonged circulation time in vivo.

Surface modification with affinity ligands capable of capturing bioactive molecules in situ is a widely used strategy for developing biofunctional materials. However, many bioactive molecules, for example zymogens, exist naturally in a “quiescent” state, and become active only when “triggered” by specific activators. In the present study, in situ activation of a surface-integrated zymogen was achieved by introducing affinity ligands for both the zymogen and its activator. Specifically a dual affinity surface was designed for the integration of plasminogen (Plg) and tissue plasminogen activator (t-PA). This surface was expected to have plasmin-generating and, therefore, fibrinolytic properties. A polyurethane surface was modified with a copolymer of 2-hydroxyethyl methacrylate and 1-adamantan-1-ylmethyl methacrylate poly(HEMA-co-AdaMA). The affinity ligands, ARMAPE peptide (for t-PA) and ε-lysine-containing β-cyclodextrin (β-CD-(Lys)7) (for Plg), were attached in sequence via covalent bonding and host–guest interactions, respectively. The resulting surfaces were shown to have high binding capacities for both t-PA and Plg while resisting nonspecific protein adsorption. Pre-loading with t-PA followed by Plg uptake from plasma generated plasmin and thus endowed the surface with fibrinolytic activity. In general the incorporation of dual affinity ligands to achieve surface-promoted bioactivity is a promising approach for the development of biofunctional materials. The method reported herein for the sequential attachment of plasminogen and t-PA affinity ligands can be extended to systems of multiple ligands generally.