Biomedical surface science: Foundations to frontiers

Adsorption of proteins from solution at the solid-liquid interface.

Surfaces containing poly(ethylene oxide) (PEO) are interesting biomaterials because they exhibit low degrees of protein adsorption and cell adhesion. In this study different molecular weight PEO molecules were covalently attached to poly(ethylene terephthalate) (PET) films using cyanuric chloride chemistry. Prior to the PEO immobilization, amino groups were introduced onto the PET films by exposing them to an allylamine plasma glow discharge. The amino groups on the PET film were next activated with cyanuric chloride and then reacted with bis-amino PEO. The samples were characterized by scanning electron microscopy, water contact angle measurements, gravimetric analysis, and electron spectroscopy for chemical analysis (ESCA). The adsorption of 125I-labeled baboon fibrinogen and bovine serum albumin was studied from buffer solutions. Gravimetric analysis indicated that the films grafted with the low-molecular-weight PEO contained many more PEO molecules than the surfaces grafted with higher-molecular-weight PEO. The high-molecular-weight PEO surfaces, however, exhibited greater wettability (lower water contact angles) and less protein adsorption than the low-molecular-weight PEO surfaces. Adsorption of albumin and fibrinogen to the PEO surfaces decreased with increasing PEO molecular weight up to 3500. A further increase in molecular weight resulted in only slight decreases in protein adsorption. Protein adsorption studies as a function of buffer ionic strength suggest that there may be an ionic interaction between the protein and the allylamine surface. The trends in protein adsorption together with the water contact angle results and the gravimetric analysis suggest that a kind of "cooperative" water structuring around the larger PEO molecules may create an "excluded volume" of the hydrated polymer coils. This may be an important factor contributing to the observed low protein adsorption behavior.

Protein adsorption to poly(ethylene oxide) surfaces.

Chapter 13 Principles underlying the role of adsorbed plasma proteins in blood interactions with foreign materials

Vitronectin--a major cell attachment-promoting protein in fetal bovine serum.

Electron spectroscopy for chemical analysis (ESCA) was used to study the surface composition of several radiation-grafted polymers in both the dry and hydrated (frozen at 160°K) states. Poly(2-hydroxyethyl methacrylate) (HEMA) and polyacrylamide, both hydrophilic polymers, were readily detected in the hydrated or dehydrated states when grafted to polyethylene substrates. For silicone rubber substrates, both grafts were observed on the hydrated surface but were significantly decreased in surface concentration upon dehydration. For grafts on a polyester-urethane, acrylamide was not a major constituent of either the dry or hydrated surface, while HEMA appeared to increase in abundance upon drying. The amount of the hydrophobic poly(ethyl methacrylate) found on the graft surface depended upon the substrate polymer used, but the surface abundance of poly(ethyl methacrylate) was not affected by drying. These results were considered in terms of polar group orientation, polymer chain mobility, substrate permeability, and the limitations of the ESCA technique. The implications of these results with respect to the use of radiation-grafted hydrophilic polymers for biomedical applications are also discussed.

Radiation-grafted hydrogels for biomaterial applications as studied by the ESCA technique.

The adsorption of proteins affects cellular interactions with foreign surfaces and thus plays an important role in determining the biocompatibility of implants. Previous studies have indicated differences in the affinity of various proteins for a given polymer, and differences in the affinity of fibrinogen for a series of polymers varying in hydrophilicity. These studies suggest that differences in the composition of the protein layer adsorbed to polymers from plasma might exist. To examine this hypothesis, the proteins adsorbed from plasma to a series of polymers varying in hydrophilicity were analyzed with the iodogram technique. Copolymers of hydroxyethyl methacrylate and ethyl methacrylate made by the radiation grafting technique were exposed to plasma for 0.5 or 150 min. The adsorbed proteins were iodinated, eluted with SDS, and separated with polyacrylamide gel electrophoresis. Fibrinogen, immunoglobulin G, hemoglobin, and a peak tentatively ascribed to prothrombin were the major proteins detected. Very little iodine was incorporated into adsorbed albumin, even though it was shown to be present by a separate experiment using dye binding. The fraction of total radioactivity associated with each of nine proteins was found to vary markedly and systematically among the surfaces. The distribution of radioactivity into the proteins was very different on 0.5 and 150-min plasma exposed polymers. The results reflect both compositional differences in the adsorbed protein layer on the polymers and differences in the accessibility of proteins to the labeling reagent in the adsorbed state. Differences in the organization of the adsorbed protein layer may play a key role in determining whether cell surface receptors can come in contact with the specific plasma protein able to further stimulate the cell.

Adsorption of proteins from plasma to a series of hydrophilic-hydrophobic copolymers. I. Analysis with the in situ radioiodination technique.

Hemoglobin adsorption to three polymer surfaces.

The composition and dynamics of the protein film adsorbed onto surfaces from blood plasma remains unknown to biomaterials development. In situ radioiodination followed by elution and electrophoretic analysis has been demonstrated as a sensitive and versatile technique for analysis of these films. Iodine-125I was incorporated into protein using iodine monochloride. After film washing, and elution with sodium dodecylsulfate (SDS), proteins were subject to SDS gel electrophoresis for MW determination. Disulfide bond reduction with dithiothreitol was also employed to detect the presence of protein subunits. Fibrinogen, albumin and IgG were readily identified when adsorbed from a single protein solution onto teflon or silicone rubber. Electrophoretic analysis of the protein layer adsorbed from plasma revealed a complex pattern of up to 8 iodinated protein species. Iodinated protein with electrophoretic behavior consistent with fibrinogen and albumin was always found after plasma exposure, but proteins of apparent MW 7-10,000; 12,000; 25,000 and others remain unidentified. Adsorbed protein patterns varied with plasma source, type of material, and time of plasma exposure.

Paul K. Weathersby

Papers

Radiation-grafted hydrogels for biomaterial applications as studied by the ESCA technique.

Adsorption of proteins from plasma to a series of hydrophilic-hydrophobic copolymers. I. Analysis with the in situ radioiodination technique.

Hemoglobin adsorption to three polymer surfaces.

A new method for analysis of the adsorbed plasma protein layer on biomaterial surfaces.

Solution stability of bovine fibrinogen