Elimination of stick-slip of elastomeric sutures by radiofrequency glow discharge deposited coatings.

Fine elastomeric sutures intended for cardiovascular surgery can exhibit "stick-slip" behavior as they are pulled through tissue; the resulting oscillatory force can damage delicate tissue or cause sutures to snap. To eliminate this undesirable effect, sutures were surface-modified using a radiofrequency glow discharge in a vapor of either hexamethyldisiloxane or hexamethyldisilazane, to produce a thin polymeric coating on the suture. The same coatings were also deposited onto aluminized tape to facilitate their characterization by measurement of air/water contact angles and by X-ray photoelectron spectroscopy. Coatings from both monomers were found to be very hydrophobic. The hexamethyldisiloxane glow discharge coatings underwent negligible oxidation when stored in air, and thus remained stable over a shelf-life period akin to what may be required of sutures. The hexamethyldisilazane glow discharge coatings, in contrast, incorporated substantial amounts of oxygen over a 3-month period. The coatings did not measurably alter the tensile properties of the sutures. The frictional properties of coated sutures were assessed by measuring the dynamic friction between the suture and ovine myocardium. Both coatings were effective in removing the inherent stick-slip behavior of polybutester sutures in this model. The coatings remained intact after several passes and proved to be robust and efficacious under various strain regimes.

Matrix-assisted laser desorption ionization mass spectrometry detection of proteins adsorbed in vivo onto contact lenses.

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect "interface conversion", in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques.

Albumin-binding surfaces: synthesis and characterization.

The nature of the proteinaceous film deposited on a biomaterial surface following implantation is a key determinant of the subsequent biological response. To achieve selectivity in the formation of this film, monoclonal antibodies have been coupled to a range of solid substrates using avidin-biotin technology. Antibody clones varied in their antigen-binding activity following insertion of biotin groups into lysine residues. Biotinylated antibodies coupled to solid substrates via an immobilized avidin bridge retained their biological activity. During immobilization of avidin a significant proportion of the protein molecules were passively adsorbed rather than covalently attached to the surface. This loosely bound material could be removed by stringent elution procedures which resulted in a surface density of 5.4 pmol avidin cm(-2). Although these conditions would be harsh enough to denature monoclonal antibodies, they did not destroy the biotin-binding activity of the residual surface-coupled avidin, enabling the subsequent immobilization of biotinylated antibodies. The two-step immobilization technique allowed the use of gentle protein modification procedures, reduced the risk of surface-induced denaturation and removed loosely bound material from the surface. The versatility of the technique encourages its application to a wide range of immobilization systems where retention of biological activity is a key requirement.

Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels.

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups ('charge') on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.

Adsorption of globular proteins on locally planar surfaces: models for the effect of excluded surface area and aggregation of adsorbed protein on adsorption equilibria.

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.

A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide.

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp 120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent attachment and non-specific binding of reactive probe molecules onto surfaces.

Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.

Roles of serum vitronectin and fibronectin in initial attachment of human vein endothelial cells and dermal fibroblasts on oxygen- and nitrogen-containing surfaces made by radiofrequency plasmas.

Fluoropolymers modified by plasma modification were studied for their suitability as surfaces for the adhesion of cells. We compared films made by plasma modification of fluoroethylenepropylene (FEP) using nitrogen-containing gases (ammonia or dimethyl acetamide) with films deposited using oxygen-containing monomers (methanol, methyl methacrylate or sequential treatment with toluene then water). The surfaces were compared for the attachment and spreading of human vein endothelial cells and human dermal fibroblasts. The initial attachment and spreading of cultured fibroblasts and endothelial cells onto films deposited using nitrogen-containing gases were equivalent to that onto films deposited using oxygen-containing monomers, but there were some differences in the mechanism of attachment. With films deposited using oxygen-containing monomers, the initial attachment and spreading of endothelial cells failed when the medium contained 15% (v/v) serum from which both fibronectin (Fn) and vitronectin (Vn) had been removed. Similarly, initial attachment and spreading of endothelial cells onto films deposited using oxygen-containing monomers were reduced by 62-86% when the cells were seeded in medium containing Vn-depleted serum (which contained Fn). Endothelial cells attached and spread onto films made using oxygen-containing monomers, when seeded in medium containing Fn-depleted serum (which contained Vn). On films deposited using nitrogen-containing gases, the adhesion of endothelial cells was only slightly reduced in Vn-depleted medium (as compared to attachment in medium containing unmodified serum). Furthermore, surfaces which had incorporated nitrogen were more effective than were oxygen-containing films in adsorbing sufficient serum Fn as to promote endothelial cell attachment. Similar results were seen for the attachment and spreading of fibroblasts as for the endothelial cells. For fibroblasts, attachment and spreading onto oxygen-containing films and onto nitrogen-containing films were not simply dependent upon either the Vn content or the Fn content of the medium. Maximal attachment and spreading of fibroblasts were, however, dependent upon adsorption of both serum Vn and Fn.

Growth of human cells on plasma polymers: putative role of amine and amide groups.

The attachment and growth of human endothelial cells and fibroblasts was studied on polymer surfaces fabricated by the polymerization of volatile amine and amide compounds in a low pressure gas plasma, and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen content of the surface layers. Infrared spectra showed the presence of amide groups, including those cases where the volatile compound ('monomer') did not contain oxygen. The performance of the surfaces in cell attachment correlated with the surface hydrophilicity and the nitrogen content, although for the latter a fair degree of scatter indicated that a more complex relationship applies. All these surfaces supported the attachment and growth of human cells. Generally, amide plasma polymers were best but the individual monomer and the plasma parameters also played a role. From comparisons of the various surfaces, it is suggested that the amide group is the main promoter of cell attachment in nitrogen-containing plasma surfaces.

Degradation of medical-grade polyurethane elastomers: the effect of hydrogen peroxide in vitro.

Treatment of Pellethane 2363-80A--a medical-grade poly(tetramethylene oxide)-based polyurethane elastomer--with 25% (w/w) hydrogen peroxide at 100 degrees C for times ranging from 24 h to 336 h led to significant decreases in ultimate tensile properties and decreases in molecular weight, both at the surface and in the bulk. IR spectral changes were similar to those observed after degradation in vivo. Differential scanning calorimetry showed that hydrogen-peroxide-induced degradation was associated with greater order in the hard domain and greater mobility in the soft domain. Studies conducted with low-molecular-weight model compounds for the hard and soft segments confirmed that methylene groups adjacent to oxygen were susceptible toward oxidation. The extent of degradation of a series of commercial polyurethanes on treatment with hydrogen peroxide (25%, 24 h, 100 degrees C) correlated well with their reported susceptibility to environmental stress cracking in vivo.

A parameterized overspeeding method for the rapid attainment of low-speed sedimentation equilibrium.

The approach of a solution of dilute, monodisperse, globular macromolecules to low-speed sedimentation equilibrium in an ultracentrifuge is simulated by numerical integration of the Lamm equation. Various combinations of overspeed time and angular velocity are used to assess the conditions needed to minimize the time it takes the solution to attain sedimentation equilibrium. The optimal overspeeding time and angular velocity are determined over a wide range of values of the molecular weight (relative molar mass) of the solute and the radial distance between the meniscus and base of the solution. The results may be expressed as simple functions which allow facile calculation of (a) the optimal overspeeding time and velocity, and (b) the time required to reach sedimentation equilibrium. The results are in reasonable agreement with previous analytical solutions which were based on several simplifying assumptions. The parameterized overspeeding procedure is shown to be robust over a wide range of conditions, and typically leads to a greater than 5-fold reduction in centrifugation time.

Indefinite isoenthalpic self-association of solute molecules.

Consideration is given to the indefinite self-association of biological molecules assuming that each step-wise addition of monomer is characterized by the same enthalpy change, and that the second virial coefficients adequately describe the thermodynamic nonideality. Statistical thermodynamic models are used to calculate translational and rotational entropy changes accompanying the addition of each monomer to the aggregate. Three polymerization schemes are explored in which the solute species are modeled as right circular cylinders of various length/radius ratios. The analysis is applied to published data on the self-association of bovine liver glutamate dehydrogenase.

Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding.

To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181-1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead-associated fluorophore with that of the cell-bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand-binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.

Isoparametric analysis of binding and partitioning processes.

An isoparametric method is described for the analysis of titration data pertaining to the binding of a ligand to an acceptor, or to the partition of a molecule between lipid and aqueous compartments. Where spectroscopic titrations are employed, the analysis does not require a priori choice of an association model, nor assumptions concerning a linear relationship between the spectroscopic signal and the amount of bound or partitioned ligand. The method has wide applicability and is illustrated with reference to (a) ligand-acceptor associations monitored by perturbation of the absorption or fluorescence signal of ligand or acceptor, and (b) cross-linking interactions such as those encountered in antigen-antibody precipitin reactions.

Analysis of competition binding assays: assessment of the range of validity of a commonly invoked assumption.

A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1-(the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms 1:1 complexes with the acceptor sites. When these conditions are satisfied, the conventional assumption is valid even if the unlabeled ligand binds to nonequivalent sites or exhibits cooperativity. Finally, we apply the theory derived for case (a) above to the binding of fluoresceinated epidermal growth factor to A431 cells and demonstrate that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.

Perturbation of phospholipid bilayers by DDT.

The localization of the effects of DDT (5-50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23 degrees C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54 degrees C or egg PC at 23 degrees C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.