Polydopamine-polyethylene glycol-albumin antifouling coatings on multiple substrates.

Aqueous-based coatings using combinations of polydopamine (PDA) (as bioadhesive) and grafted polyethylene glycol (PEG) (as antifouling agent) have been reported to reduce biofouling on multiple material surfaces. However, the achievable PEG grafting density and antifouling performance are limited, leaving exposed PDA to provide sites for attachment of proteins and cells. In the present work, we investigate the polymerization of dopamine on three substrate materials, polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass, to evaluate the utility of the PDA coatings for application to multiple materials. Additionally, we propose that the PDA-PEG method may be improved by "backfilling" with bovine serum albumin (BSA) as a blocker covering exposed PDA. AFM and ellipsometry studies revealed substantial differences in PDA thickness and roughness on each material despite their being modified under the same conditions. X-ray photoelectron spectroscopy (XPS) and water contact angle data revealed differences in PEG grafting on these materials as a consequence of varying PDA surface roughness, with the highest PEG coverage achieved on PC-PDA surfaces of intermediate roughness and lower PEG attachment on smoother PDMS-PDA surfaces. Fibrinogen adsorption experiments showed significantly less fouling on PDA-BSA surfaces compared to PDA-PEG for all three substrates, the larger BSA molecules presumably providing greater coverage of the PDA. On the PC and PDMS substrates, backfilling the PDA-PEG surfaces with BSA gave significant reductions in fibrinogen adsorption, with the lowest adsorption of 75 ng cm-2 achieved on PC-PDA-PEG/BSA.

Interactions of Apolipoproteins AI, AII, B and HDL, LDL, VLDL with Polyurethane and Polyurethane-PEO Surfaces.

The lipoproteins (HDL, LDL, VLDL) are important components of blood present in high concentration. Surprisingly, their role in blood-biomaterial interactions has been largely ignored. In previous work apolipoprotein AI (the main protein component of HDL) was identified as a major constituent of protein layers adsorbed from plasma to biomaterials having a wide range of surface properties, and quantitative data on the adsorption of apo AI to a biomedical grade polyurethane were reported. In the present communication quantitative data on the adsorption of apo AI, apo AII and apoB (the latter being a constituent of LDL and VLDL), as well as the lipoprotein particles themselves (HDL, LDL, VLDL), to a biomedical segmented polyurethane (PU) with and without an additive containing poly(ethylene oxide) (material referred to as PEO) are reported. Using radiolabeled apo AI, apo AII, and apoB, adsorption levels on PU from buffer at a protein concentration of 50 µg/mL were found to be 0.34, 0.40, and 0.14 µg/cm(2) (12, 23, and 0.25 nmol/cm(2)) respectively. Adsorption to the PEO surface was <0.02 µg/cm(2) for all three apolipoproteins demonstrating the strong protein resistance of this material. In contrast to the apolipoproteins, significant amounts of the lipoproteins were found to adsorb to the PEO as well as to the PU surface. X-ray photoelectron spectra, following exposure of the surfaces to the lipoproteins, showed a strong phosphorus signal, confirming that adsorption had occurred. It therefore appears that a PEO-containing surface that is resistant to apolipoproteins may be less resistant to the corresponding lipoproteins.

Non-fouling biomaterials based on blends of polyethylene oxide copolymers and polyurethane: simultaneous measurement of platelet adhesion and fibrinogen adsorption from flowing whole blood.

Measurements of platelet adhesion and fibrinogen adsorption from flowing whole blood to a series of polyethylene oxide (PEO)-based materials were carried out. A unique experimental design was used in which both quantities were measured in the same experiment. The materials consisted of a polyurethane (PU) as a matrix into which various triblock copolymers of general structure PEO-PU-PEO were blended; the PU block was the same in all materials but the PEO blocks ranged in molecular weight from 550 to 5000. Platelets were isolated from fresh human blood and labeled with (51)Cr; purified fibrinogen was labeled with (125)I. A whole blood preparation containing these labeled species was used for the adhesion/adsorption studies. The surfaces were exposed to the flowing blood in a cone and plate device at a wall shear rate of 300 s(-1). It was found that both platelet adhesion and fibrinogen adsorption decreased with increasing copolymer content in the blends and with decreasing PEO block size for a given copolymer content. The block size effect was due probably to higher PEO surface coverage for the lower molecular weight blocks. Fibrinogen adsorption and platelet adhesion were linearly and strongly correlated. The best performing materials showed very low fibrinogen adsorption of the order of 25 ng/cm(2), and correspondingly low platelet densities around 10,000 per cm(2), i.e. fractional platelet coverage in the vicinity of 0.2%.

Platelet Adhesion and Fibrinogen Accretion on a Family of Elastin-Like Polypeptides.

Previous work in our laboratory showed the potential of using a human recombinant elastin-like polypeptide (ELP) as a thromboresistant coating. In this work we investigate the use of three particular ELPs (ELP1, ELP2 and ELP4), that differ by molecular weight and number of repeating hydrophobic and cross-linking domains, as coatings to improve blood-contacting properties. All three ELPs were passively adsorbed on Mylar surfaces. Differences in water contact angle and surface concentration were found among the three ELP coatings, with the shortest polypeptide, ELP1, being the most hydrophilic and abundant on the surface (55°, 0.76 µg/cm(2)), followed by ELP2 (55°, 0.35 µg/cm(2)) and ELP4, the longest of the three (66°, 0.25 µg/cm(2)), respectively. The blood interactions of the ELP coatings were investigated by measuring fibrinogen adsorption and platelet adhesion in whole blood under laminar flow in a cone and plate viscometer configuration. In general, platelet adhesion to the ELP-coated surfaces was found to correlate with fibrinogen adsorption. Decreases in fibrinogen accretion and platelet adhesion were observed for ELP-coated compared to uncoated surfaces. The magnitude of the decreases was found to depend on the ELP sequence length, with ELP4 exhibiting the lowest levels of fibrinogen adsorption and platelet adhesion at 43 ± 24 ng/cm(2) and 113 ± 77 platelets/mm(2), respectively.

Nonfouling biomaterials based on polyethylene oxide-containing amphiphilic triblock copolymers as surface modifying additives: adsorption of proteins from human plasma to copolymer/polyurethane blends.

Three polyethylene oxide-polyurethane-polyethylene oxide (PEO-PU-PEO) block copolymers of variable PEO block size (MW 550, 2000, and 5000) were used to modify the surface of a conventional segmented polyurethane (PU) with the objective of inhibiting interactions with proteins. The surface-active copolymers were blended with the PU by solution methods. Protein adsorption from human plasma to the modified materials was investigated using radiolabeling and immunoblotting methods. From the radiolabeling experiments, it was found that fibrinogen adsorption from plasma to all of the modified surfaces was much lower than to the unmodified PU matrix. For blends of low copolymer content, resistance to adsorption was greatest on the copolymer 1 (PEO550)-modified materials, and increased with increasing copolymer content for all three blend types. At high copolymer content inhibition of adsorption was very strong and independent of PEO block size. The immunoblotting experiments showed that on materials of high copolymer content (20 wt %), the proteins investigated (fibrinogen, albumin, complement C3, and apolipoprotein A-I) were undetectable. At low copolymer content (< or = 5 wt %), the blends of copolymer 1, with the shortest PEO block, exhibited greater protein resistance than those of copolymers 2 and 3 (PEO blocks of MW 2000 and 5000, respectively), and resistance decreased with decreasing protein size. Evidence of complement activation was seen for the blends of low copolymer content. Adsorption of C3 and complement activation decreased with increasing content of the copolymers. It was concluded that surface density of PEO is more important than chain length for protein resistance in contact with plasma.

Surface modifications of Nitinol for biomedical applications.

Cathodic electrophoretic deposition (EPD) has been utilized for the fabrication of composite films for the surface modification of NiTi shape memory alloys (Nitinol). In the proposed method, chitosan (CH) was used as a matrix for the incorporation of other functional materials, such as heparin, hydroxyapatite and bioglass. Chitosan-heparin films were deposited from solutions of non-stoichiometric chitosan-heparin complexes. It was found that the addition of anionic heparin to the solutions of cationic chitosan resulted in a significant increase in the cathodic deposition rate. The thickness of the films prepared by this method varied in the range of 0.1-3 microm. The ability of the chitosan-heparin films to bind antithrombin, as measured by binding of (125)I-radiolabeled antithrombin, was much greater than that of pure chitosan films. Composite chitosan-hydroxyapatite films, with thickness of 1-30 microm, were obtained as monolayers or laminates, containing chitosan-hydroxyapatite layers, separated by layers of pure chitosan. The hydroxyapatite nanoparticles showed preferred orientation in the chitosan matrix with the c-axis parallel to the substrate surface. The films showed corrosion protection of the Nitinol substrates in Ringer's physiological solutions. The feasibility of the fabrication of composite films containing hydroxyapatite and bioglass in the chitosan matrix has been demonstrated. The method offers the advantages of room temperature processing. The deposition mechanisms and possible applications of the films are discussed.

Nonfouling biomaterials based on polyethylene oxide-containing amphiphilic triblock copolymers as surface modifying additives: protein adsorption on PEO-copolymer/polyurethane blends.

Surface modification of a segmented polyurethane was achieved by blending with novel PEO-containing amphiphilic triblock copolymers (PEO-polyurethane-PEO). Three copolymers having different PEO MW (550, 2000, 5000) were used as surface modification additives. The protein resistance of the blend surfaces was evaluated using radiolabeling methods. On the blends of copolymers with PEO blocks of MW 2000 and 5000, fibrinogen adsorption from physiologic buffer decreased with increasing copolymer content up to 20 wt%. On the blends with PEO blocks of MW 550, resistance to adsorption for a given copolymer content was much greater. For all three blend types at 20% copolymer content, reductions in adsorption compared to the unmodified PU matrix were greater than 95%. Reductions in adsorption were similar for the 20% blends and surfaces prepared by coating the copolymers directly on the matrix, suggesting that the 20% blend surfaces were completely covered by copolymer. At low copolymer content (< or =10 wt %), fibrinogen adsorption decreased with decreasing PEO block length. This was probably due to increasing surface coverage of the copolymers with decreasing block length. It is therefore concluded that surface density of PEO is more important than PEO MW for the protein resistance of these surfaces. Lysozyme, a much smaller protein, showed adsorption trends similar to fibrinogen. The adsorption of fibrinogen and lysozyme from binary solutions to blends of the copolymer with PEO blocks of 2000 MW was investigated to probe the effects of protein size on adsorption resistance. Fibrinogen and lysozyme showed similar fractional decreases in adsorption relative to the PU matrix independent of the surface density of PEO. However lysozyme was enriched in the surface relative to the solution, that is, it was adsorbed preferentially to fibrinogen.

Nonfouling biomaterials based on polyethylene oxide-containing amphiphilic triblock copolymers as surface modifying additives: solid state structure of PEO-copolymer/polyurethane blends.

Three novel polyethylene oxide-containing amphiphilic triblock copolymers (PEO-PU-PEO) with PEO MWs 550, 2000, and 5000, were blended with a segmented polyurethane (PU). It was expected that the block copolymers would act as surface modifiers to produce surfaces rich in PEO. The solid state properties of the PEO-copolymer/PU blends were studied by infrared spectroscopy, differential scanning calorimetry, and tensile stress-strain measurements. Infrared analysis showed no significant hydrogen bonding between the PEO blocks of the copolymers and the PU matrix. Differential scanning calorimetry data indicated that for copolymer content up to 5 wt % the microphase structure of the blends was indistinguishable from that of the unmodified PU matrix; for copolymer content of 10% or greater, the blends showed phase separated structures. Similarly the tensile stress-strain properties of the blends were essentially the same as those of the matrix up to 5 wt % copolymer. At higher copolymer content, however, the tensile strength decreased with increasing content of the copolymers; for a given copolymer content the change in tensile properties increased with increasing PEO MW. The structures of the 20% blends were also investigated after extraction with toluene (copolymers soluble, matrix insoluble). Bulk compositional change upon extraction was determined by nuclear magnetic resonance spectroscopy. Surface compositional change was studied by X-ray photoelectron spectroscopy and water contact angles. Surface morphology was observed using scanning electron microscopy and atomic force microscopy. It was shown that the copolymers were removed from the blends by extraction and that the extent of removal increased with decreasing MW of the PEO block. After toluene extraction, the blend surfaces showed advancing water contact angle and surface elemental composition similar to those of the PU matrix. However in contrast to the relatively smooth matrix surface, the extracted blend surfaces were "decorated" with lacunae or pits. Consistent with the weight loss trends, the extent of pitting was greater for the copolymers having shorter PEO blocks, suggesting that surface enrichment of the copolymers increased with decreasing MW of the copolymers.

Fibrinolytic properties of lysine-derivatized polyethylene in contact with flowing whole blood (Chandler loop model).

This article reports on the concept of a fibrinolytic surface based on the preferential adsorption of endogenous plasminogen from blood. Data are presented indicating that such a surface, when pretreated with tissue-type plasminogen activator (tPA), is able to dissolve nascent thrombus generated in contact with flowing whole blood. Polyethylene (PE) surfaces were modified by attaching a lysine-containing polymer using photochemical methods as reported previously (McClung et al., J Biomed Mater Res 2000;49:409-414). The lysine residues were bound chemically to the polymer via the alpha-amino groups leaving the epsilon-amino groups free (epsilon-Lys surface). Control surfaces were (a) unmodified PE, (b) PE modified with the coating polymer containing no lysine, and (c) PE modified with the polymer containing lysine bound via the epsilon-amino group. The materials in tubing form were evaluated in contact with nonanticoagulated flowing human whole blood in a modified Chandler Loop experiment. They were first treated with tPA to allow activation of adsorbed plasminogen to plasmin. It was found that thrombus formation was initiated within 15-25 min (depending on donor blood) on all surfaces, as indicated by the formation of platelet aggregates. On the controls (including the lysine-containing material in which the epsilon-amino group was used in the binding reaction) thrombogenesis continued till the tubing was occluded and blood flow ceased. On the epsilon-Lys surface, thrombogenesis was interrupted at various stages depending on the donor blood; in all cases any thrombus generated was dissolved within minutes. It was shown that thrombolysis was due to the fibrinolytic action of plasmin generated at the surface and not to plasmin formed by traces of tPA released into the blood. This work provides further evidence of the efficacy of this approach to the development of a fibrinolytic surface.

Fibrinogen adsorption and platelet lysis characterization of fluorinated surface-modified polyetherurethanes.

A polyetherurethane (PU) was modified using fluorinated surface-modifying macromolecules (SMMs). A double radiolabel method was used simultaneously to measure the number of adhered platelets ((51)Cr) and the quantity of adsorbed Fg ((125)I), in a cone-and-plate instrument. The objectives were to determine if adsorbed Fg levels correlated to platelet adhesion on the surfaces, and to assess if any reductions in platelet adhesion for the SMM-treated surfaces resulted from surface-induced platelet lysis, rather than changes directly related to lower platelet activation and attachment on the novel surfaces. Platelet lysis was determined from lactate dehydrogenase (LDH) and unbound (51)Cr released into plasma isolated from whole blood exposed to test materials. The corresponding Fg adsorption, evaluated under the same platelet adhesion conditions, did not account for the reduced platelet adhesion on the treated surfaces. LDH and (51)Cr platelet release were very low and indicated no statistically significant differences between the materials. It was therefore concluded that platelet lysis did not contribute to the reduction in platelet adhesion characteristic observed on the SMM-treated surfaces. More importantly, the work emphasizes that the platelet activation cannot be inferred to by assessing the quantity of fibrinogen as is commonly done in the literature. The finding suggests a much more complex mechanism of action for the SMM surface modifiers. On-going work is investigating other Fg parameters such as protein binding affinity and protein conformational state in order to establish the mechanism by which the fluorinated surface modifiers may be reducing platelet adhesion via intermediary changes in initial protein adsorption.

Fibrinogen surface distribution correlates to platelet adhesion pattern on fluorinated surface-modified polyetherurethane.

In previous work, it had been shown that platelet adhesion could be reduced by fluorinating surfaces with oligomeric fluoropolymers, referred to as surface-modifying macromolecules (SMMs). In the current study, two in vitro blood-contacting experiments were carried out on a polyetherurethane modified with three different SMMs in order to determine if altered platelet adhesion levels could be related to the pattern of adsorbed protein and more specifically to the manner in which fibrinogen (Fg) distribution occurs at the surface. In the first experiment, the materials were placed in whole human blood and the adherent platelets were viewed with high-resolution scanning electron microscopy (SEM). In a second experiment, the materials were incubated with human plasma with the absence of platelets. The plasma contained 5% fluorescent-Fg. The materials were then viewed with a fluorescence microscope and images were collected to define the distribution of high-density fluorescent-Fg areas. The SEM and fluorescent-Fg images were imported to Image Pro Plus imaging software to measure the area, length and circularity and a bivariate correlation test was conducted between the two sets of data. For area and length morphology parameters, there were high and significant correlations (r > 0.9, p < 0.05) between the platelets and Fg aggregates. The data suggest that the Fg distribution may serve as a predictor of platelet morphology/activation and provides insight into the non-thrombogenic character of biomaterials containing the fluorinated SMMs.

Interactions of fibrinolytic system proteins with lysine-containing surfaces.

Studies on the interactions of tissue plasminogen activator (tPA) and plasminogen with polyurethane surfaces containing epsilon-lysine moieties (epsilon-amino group free) are reported. These surfaces are considered to have the potential to dissolve nascent clots that may be formed on them. For adsorption from both single protein solutions and plasma, the surfaces were found to have a high capacity for tPA as well as plasminogen. A significant fraction of preadsorbed tPA was displaced from the epsilon-lysine surfaces upon contact with plasma. These surfaces, when preadsorbed with tPA and then incubated with plasma, were able to dissolve incipient clots formed around them. However, the clot-dissolving capacity diminished as the time of plasma incubation increased, presumably due to loss of tPA. It was also shown that in plasma, preadsorbed tPA is displaced from these surfaces largely by plasminogen, which thus appears to have a greater binding affinity than tPA for the epsilon-lysine moieties. Finally, it was found that in plasma, the epsilon-lysine surfaces interact with plasminogen in a dynamic manner, and that about 70% of the bound plasminogen is exchanging continuously with plasminogen in the plasma.

The influence of protein adsorption and surface modifying macromolecules on the hydrolytic degradation of a poly(ether-urethane) by cholesterol esterase.

Previous investigations have demonstrated that the inflammatory cell derived enzyme, cholesterol esterase (CE) could degrade polyurethanes (PUs) by hydrolyzing ester and urethane bonds. Studies that have investigated the development of protective coatings for PUs have reported that the polymer degradation of polyester-urethanes (PESUs) can be reduced with the use of fluorine containing surface modifying macromolecules (SMMs). Since these latter studies were carried out in the presence of relatively pure enzyme, it has not been shown if SMMs would still provide an enhanced inhibitory effect if surfaces were pre-exposed to plasma proteins. This would be more representative of the in vivo scenario since protein adsorption would occur before the appearance of monocyte-derived macrophages which would be a primary source of esterase activities. The current investigation has focused on studying the influence of fibrinogen (Fg) as a simple model of protein adsorption in order to assess the effect of CE in combination with protein on polyether-urethane (PEU) surfaces. The materials were prepared with and without SMMs, and were pre-coated with Fg prior to carrying out biodegradation studies. The pre-adsorption of Fg onto the modified and non-modified surfaces provided a significant delay in the hydrolytic action of CE onto the PEU substrates. However, the effect was gone by 70 days and by the 126th day of incubation, both Fg coated and non-Fg coated groups had the same level of degradation. The difference between Fg coated and non-coated substrates was much smaller for materials containing SMMs. In addition, the pre-adsorption of Fg did not alter the SMMs' ability to provide a more biostable surface over the 4 month incubation period.

Deterioration of polyamino acid-coated alginate microcapsules in vivo.

The implantation of immuno-isolated recombinant cell lines secreting a therapeutic protein in alginate microcapsules presents an alternative approach to gene therapy. Its clinical efficacy has recently been demonstrated in treating several genetic diseases in murine models. However, its application to humans will depend on the long-term structural stability of the microcapsules. Based on previous implantations in canines, it appears that survival of alginate-poly-L-lysine-alginate microcapsules in such large animals is short-lived. This article reports on the biological factors that may have contributed to the degradation of these microcapsules after implantation in dogs. Alginate microcapsules coated with poly-L-lysine or poly-L-arginine were implanted in subcutaneous or intraperitoneal sites. The retrieved microcapsules showed a loss of mechanical stability, as measured by resistance to osmotic stress. The polyamino acid coats were rendered fragile and easily lost, particularly when poly-L-lysine was used for coating and the intraperitoneal site was used for implantation. Various plasma proteins were associated with the retrieved microcapsules and identified with western blotting to include Factor XI, Factor XII, prekallikrein, HMWK, fibrinogen, plasminogen, ATIII, transferrin, alpha-1-antitrypsin, fibronectin, IgG, alpha-2-macroglobulin, vitronectin, prothrombin, apolipoprotein A1, and particularly albumin, a major Ca-transporting plasma protein. Complement proteins (C3, Factor B, Factor H, Factor I) and C3 activation fragments were detected. Release of the amino acids from the microcapsule polyamino acid coats was observed after incubation with plasma. indicating the occurrence of proteolytic degradation. Hence, the loss of long-term stability of the polyamino acid-coated alginate microcapsules is associated with activation of the complement system, degradation of the polyamino acid coating, and destabilization of the alginate core matrix, probably through loss of calcium-mediated ionic cross-linking of the guluronic acid polymers in the alginate. These destructive forces may be slightly mitigated by using poly-L-arginine instead of poly-L-lysine for coating and by implanting in a subcutaneous instead of an intraperitoneal site. However, the long-term stability of such devices may require significant improvements in the microcapsule polymer chemistry to withstand such biological impediments.

Towards practical soft X-ray spectromicroscopy of biomaterials.

Scanning transmission X-ray microscopy (STXM) is being developed as a new tool to study the surface chemical morphology and biointeractions of candidate biomaterials with emphasis on blood compatible polymers. STXM is a synchrotron based technique which provides quantitative chemical mapping at a spatial resolution of 50 nm. Chemical speciation is provided by the near edge X-ray absorption spectral (NEXAFS) signal. We show that STXM can detect proteins on soft X-ray transparent polymer thin films with monolayer sensitivity. Of great significance is the fact that measurements can be made in situ, i.e. in the presence of an overlayer of the protein solution. The strengths, limitations and future potential of STXM for studies of biomaterials are discussed.

Fluorinated surface-modifying macromolecules: modulating adhesive protein and platelet interactions on a polyether-urethane.

Polyether-urethanes (PEUs) have been the materials of choice for the manufacture of conventional blood-contacting devices. Nevertheless, biostability and blood compatibility are still among the principal limitations in their long-term application. Studies investigating the development of protective coatings for PEUs have shown that degradation can be reduced with the use of fluorinated surface-modifying macromolecules (SMMs). It has also been hypothesized that SMM-modified PEU surfaces may exhibit improved blood compatibility because other studies have shown a modulation in fibrinogen adsorption onto these surfaces. To determine the blood compatibility of a PEU-containing fluorinated SMMs, a series of in vitro experiments were designed to study the pattern of protein adsorption from plasma and then to assess the nature of platelet adhesion and activation on each substrate. Western blot analysis as well as single protein studies revealed that the dominant "adhesive proteins" [fibrinogen (Fg), fibronectin (Fnc), and vitronectin (Vnc)] were adsorbed on two of the SMM-containing PEUs in lower amounts relative to unmodified base. Platelet adhesion and activation data further highlighted the differences among the various substrates. It was shown that the unmodified base had a higher number of adhered platelets relative to the SMM-modified surfaces, and that of the SMM-containing substrates, which showed the lowest levels of adhesive proteins also, exhibited significantly lower platelet densities. Close morphological examination further revealed that platelets residing on these latter substrates were not appreciably activated. Based on the current evidence, it is believed that the fluorinated SMMs demonstrate good potential for the development of surfaces with minimal thrombogenic character in in vivo applications.

Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma.

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.

Lysine-derivatized polyurethane as a clot lysing surface: conversion of adsorbed plasminogen to plasmin and clot lysis in vitro.

Polyurethane surfaces to which lysine residues are immobilized by photochemical methods are proposed as a basis for clot lysing surfaces. The lysines are attached in such a way that the epsilon-amino and carboxyl groups are free. We showed previously that these surfaces, when placed in contact with plasma, adsorb only plasminogen and virtually no other proteins (McClung et al., J. Biomed. Mater. Res. 49 (2000) 409). In this communication, data based on a chromogenic substrate assay are presented showing that plasminogen adsorbed to these surfaces is readily converted to plasmin in the presence of tissue-plasminogen activator (t-PA). Moreover, the rate of activation on the surface is considerably greater than in solution. Experiments demonstrating the ability of these surfaces to dissolve fibrin clots are also reported. Surfaces exposed to plasma and then to t-PA were placed in citrated plasma. On recalcification, clotting was initiated, but the incipient clots were soon dissolved. On control surfaces (no lysine or lysine in which the epsilon-amino groups were not available) coagulation continued until a stable clot was formed. Similar observations were made when the plasma/t-PA exposed surfaces were placed in a pure fibrinogen solution and thrombin was added.

Exploiting the current paradigm of blood-material interactions for the rational design of blood-compatible materials.

The paradigm of tissue material interactions, which holds that protein adsorption is the first event following contact and determines the later interactions of cells, is invoked to propose a design strategy for biocompatibility. Control of protein interactions is the key element, and it is suggested that nonspecific protein adsorption must be prevented while the adsorption of specific proteins that are expected to result in appropriate bioactivity must be promoted. Modification with polyethylene oxide has been investigated extensively as a means of preventing nonspecific adsorption. Examples of proteins that could be targeted for specific adsorption are antithrombin III to prevent coagulation and albumin to minimize platelet adhesion. Two examples of surfaces designed for specific adsorption from the author's laboratory are discussed: the incorporation of thrombin binding peptides to give a thrombin scavenging surface, and the incorporation of lysine to give a plasminogen specific surface with the potential to dissolve clots.

Adsorption of plasminogen from human plasma to lysine-containing surfaces.

The objective of this work is to develop blood-contacting surfaces that will dissolve nascent clots that may begin to form on them. Surfaces were prepared consisting of a polyurethane to which a coating reagent was attached covalently by photochemical methods. The coating reagent was a polyacrylamide with lysine and benzophenone (for photochemical attachment) moieties pendant to the chains. It was hypothesized that via the lysine moieties such surfaces would show specific binding affinity for plasminogen, the principal component of the fibrinolytic system in blood. Surfaces of varying lysine content in which the lysine was bound through the alpha-amino groups, leaving the epsilon-amino groups free, were investigated. A control surface in which the lysine was bound through the epsilon-amino groups was also examined. Advancing water contact angles showed the surfaces to be hydrophilic. Hydrophilicity was found to decrease as the lysine content increased. Adsorption of plasminogen from plasma was studied using radioiodinated plasminogen as a tracer. For the epsilon-lysine surfaces, adsorption increased with increasing lysine content and reached a value of 1.2 microg/cm(2) for the surface with the highest lysine content, that is, in the range expected for a compact monolayer of plasminogen. The control surfaces, which contained either no lysine or lysine in which the epsilon-amino groups were unavailable, adsorbed very small amounts of plasminogen. Immunoblots were obtained for the proteins eluted from the surfaces after incubation with plasma. For the control surfaces, most of the proteins tested for (some 20 in all) were present. However, for the surface containing the highest concentration of epsilon-lysine, only plasminogen was detected in a significant amount. It is concluded that the epsilon-lysine surface adsorbs plasminogen to the exclusion of the other plasma proteins. Studies to examine the fibrinolytic properties of these surfaces will constitute the next phase of this work.