Karin D. Caldwell

Surface modification for controlled studies of cell-ligand interactions.

This work describes a method for coupling cell adhesion peptides to hydrophobic materials for the purpose of controlling surface peptide density while simultaneously preventing nonspecific protein adsorption. PEO/PPO/PEO triblock copolymers (Pluronic F108) were equipped with terminal pyridyl disulfide functionalities and used to tether RGD containing peptides to polystyrene (PS). The density of F108 on PS was 1.4 E5 +/- 2.12 E1 molecules/microm2. XPS and ToF SIMS indicated that the F108 coating was homogeneous and that the unmodified and activated F108 distributed evenly on PS. By mixing unmodified F108 with PDS-activated F108 prior to adsorption, it was possible to vary peptide density between 0 and 8.7 E4 +/- 2.66 E3 peptides/microm2, while otherwise, maintaining consistent surface properties. GRGDSY grafted PS supported cell attachment, spreading, and development of cytoskeletal structure, all of which were found to increase with increasing peptide density. Cell proliferation followed this same trend, however, maximal growth occurred at a submaximal peptide density. Cell aspect ratio varied in a biphasic manner with GRGDSY density. F108 coated PS and GRGESY grafted PS were inert to cell adhesion. Cells released from GRGDSY grafted PS upon addition of either a reducing agent or free GRGDSY, which indicates that cell-substrate interactions were mediated solely by the tethered peptides.

A novel method for surface modification to promote cell attachment to hydrophobic substrates

The ability to study and regulate cell behavior at a biomaterial interface requires strict control over material surface chemistry. Perhaps the greatest challenge to researchers working in this area is preventing the fouling of a given surface due to uncontrolled protein adsorption. This work describes a method for coupling peptides to hydrophobic materials for the purpose of simultaneously preventing nonspecific protein adsorption and controlling cell adhesion. A hexapeptide containing the ubiquitous RGD cell-adhesion motif was coupled to polystyrene (PS) via a polyethylene oxide (PEO) tether in the form of a modified PEO/PPO/PEO triblock copolymer. Triblocks were adsorbed onto PS at a density of 3.3 +/- (5.14 x 10(-4)) mg/m2 (1.4 x 10(5) +/- 2.12 x 10(1) molecules/microm2), which was determined by isotope 125I labeling. The peptide, GRGDSY, was activated at the N terminus with N-Succinimidyl 3-(2-pyridyldithio) propionate and coupled to immobilized triblocks where the terminal hydroxyls had been converted to sulfhydryl groups. Surface peptide density was measured by amino acid analysis and found to be 1.4 x 10(4) +/- 0.47 x 10(4) molecules/microm2. PS modified with PEO/PPO/PEO copolymers alone was found to be inert to cell adhesion both in the presence of serum proteins and when exposed to activated RGD peptide. In contrast, PS conjugated with RGD via endgroup-activated PEO/PPO/PEO copolymers supported cell adhesion and spreading. The surface coupling scheme reported here should prove valuable for studying cell-ligand interactions under simplified and highly controlled conditions.

Electrical Field-Flow Fractionation of Proteins

Protein separation has been achieved by electrical field-flow fractionation, a heretofore unrealized separation technique. Some advantages of this method relative to electrophoresis are the low voltage required, the lack of adverse heating and support effects, and the existence of the method as an elution technique. A comparison of theoretical and experimental retention shows good agreement.

Observations on Anomalous Retention in Steric Field-Flow Fractionation

Abstract We discuss two anomalies that have characterized our recent work in steric field-flow fractionation (steric FFF): (a) retention has been found to vary with flow rate, contrary to simple theory; and (b) low density cells elute earlier than theory predicts. After reviewing the theory, we describe experiments in both gravitational and centrifugal fields applied to two spherical beads (microporous silica and polystyrene latex) of essentially equal sizes but different densities. The velocity dependence of retention is verified for both kinds of particles, but we find that retention depends dramatically on density as well. We speculate that these anomalous dependencies originate in a velocity-dependent lift force acting to pull the particle away from the wall. This hypothesis is supported by the observation that an increase in field strength increases retention. We conclude that a controllable field strength (as in a centrifuge) is an important asset in steric FFF, permitting increased separation speed...

Colloid characterization by sedimentation field-flow fractionation: I. Monodisperse populations

This paper initiates a series of reports intended to develop, refine, and evaluate the methodology of sedimentation field-flow fractionation (sedimentation FFF) applied to the characterization of colloidal materials. Particle parameters subject to characterization include effective mass, actual mass, density, polydispersity, diffusivity, and various particle dimensions. Monodisperse particle populations are the focus of this paper; size distribution and other studies will follow. The general advantages of sedimentation FFF noted for such studies include the theoretically predictable characteristics of the method, experimental flexibility and redundancy, use of nearly any desired suspending medium, small (<1 mg) sample requirements, high resolution, and fraction collection for additional characterization. For monodisperse populations, it is shown that both retention and peak dispersion measurements lead to the various mass and dimensional parameters noted above. The fundamental theory of retention and peak dispersion is summarized; various equations are then derived showing how particle diameter and other particle parameters relate to retention and peak dispersion measurements. Experimental data are reported for five polystyrene latex beads in the nominal diameter range 0.220−0.945 μm measured in two sedimentation FFF systems. Both rigorous and approximate procedures for calculating particle diameter d were divided into three categories: those based on retention measurements (R) only, those based on peak dispersion or plate height (H) only, and those based on a combination (RH) of the two measurements. Analysis of the results suggests that d obtained from rigorous R methods are accurate to about 1–3%. However, it appears that measured peak width may be ∼20% too large, giving d values ∼5% too small by H methods and ∼40–50% too large by RH methods. Various approximations for R, H, and RH methods are evaluated. In another direction, polydispersity values appear accurate to within 1% or better. However, particle density calculated using a combination of retention and peak dispersion data is in error by a factor of ∼3, in contrast to the ∼0.002 g/cm3 accuracy yielded by retention measurements.

Mucin Coating on Polymeric Material Surfaces to Suppress Bacterial Adhesion

Abstract Mucin, a group of large glycoproteins, constitutes one of the major components of mucous which covers the lumenal surfaces of epithelial organs and serves as a physical barrier between the extracellular milieu and the plasma membrane. The molecules have a generic structure consisting of a thread-like peptide backbone with densely packed carbohydrate side chains. Protein and carbohydrate contents are about 30 and 50%, respectively. On hydrophobic materials in aqueous environments the naked parts of mucin’s protein backbone will adhere due to their hydrophobicity, while the carbohydrate side chains are thought to orient themselves away from the surface. This gives the mucin molecules their unique properties as surfactants, i.e. they tend to adsorb to hydrophobic surfaces via protein-surface interactions while they hold water molecules via their hydrophilic oligosaccharide clusters. In the present work, bovine submaxillary gland mucin (BSM) is purified by SEC and subsequently characterized with PAGE. Four polymeric materials, PMMA, silicone, Tecoflex® polyurethane and polystyrene, are selected as coating targets. Contact angle measurements show significant changes in these materials after coating with BSM. Surface concentrations of adsorbed BSM are determined by amino acid analysis and found to correlate well with observed reductions in contact angle. Both Staphylococcus aureus and CNS S. epidermidis are used to contaminate uncoated and BSM coated surfaces of all four materials, demonstrating a correlation between suppression of bacterial adhesion and surface concentration of BSM. Thus, bacterial counts on the coated PMMA, PS, PU and silicone specimens amount to ≈3, 10, 8 and 30% of the counts found on their uncoated counterparts. These results suggest that mucin coatings could profitably be employed to reduce the risk of microbial infections on polymeric biomaterials.

Separation of Human and Animal Cells by Steric Field-Flow Fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.

Colloid characterization by sedimentation field-flow fractionation: II. Particle-size distribution

Abstract It is shown that the accurate theoretical relationship between particle size and retention volume in sedimentation field-flow fractionation, as established in the first paper (1) of the series, provides a raw size or mass distribution spectrum directly from each experimental run. The advantages of this approach to obtaining particle-size distribution curves include the theoretical tractability which obviates the need for calibration standards, the controllable centrifugal field strength which provides flexibility and redundancy, the high resolution, and the capability for collecting narrow fractions for additional characterization. The final size distribution curve is obtained by applying two corrections to the fractogram: one for change of scale and one for detector response. In order to test the methodology, experiments are described using polyvinyl chloride latex beads in the diameter range 0.1−0.4 μm. The effects of the two corrections are shown. In order to test self-consistency under a variety of experimental conditions, size distribution curves are compared in which there are systematic variations in sample size, field strength, and flow velocity. The agreement is excellent in all cases. Finally, size distribution curves obtained by sedimentation FFF, transmission electron microscopy (TEM), and disk centrifugation are compared. The first two are in good agreement if one assumes a reasonable bead shrinkage (∼22%) in the electron beam during the TEM procedure.

Steric field-flow fractionation as a tool for the size characterization of chromatographic supports

Abstract Steric field-flow fractionation is described andd its application to the determination of the mean size and size distribution of chromatographic supports is suggested. After measuring systematic departures from theoretical retention values, a calibration curve is constructed using microporous silica beads whose mean diameters (4.4, 6.4, 12.1 μm) are accurately determined by electron microscopy. Other spherical chromatographic support materials are shown to cluster around the calibration curve. Fractograms (elution curves) are then shown for 15 spherical supports and six irregular supports. It is shown that the fractograms yield information on mean particle diameters, dispersion, and skewness, the latter possibly reflecting high concentrations of fine and coarse particles. Some limitations and future needs in the application of the method are noted.

Plasma protein interactions with Pluronic™-treated colloids

Abstract Hydrophobic polystyrene colloids are known to rapidly take up numerous proteins from solution, including the key plasma proteins albumin and fibrinogen. However, when coated with poly(ethylene oxide) (PEO)-containing block copolymers these particles become much less prone to protein adsorption. The noted protein repulsion is thought to be influenced by three important factors: the surface concentration of the adsorbed polymers, the thickness of the adsorbed layer and the surface mobility of the polymer chains. At a given surface concentration, the increase in both thickness and dynamics is directly correlated with a decrease in protein adsorption, as seen from the interactions of selected copolymer adsorption complexes with human fibrinogen. The significant suppression of fibrinogen adsorption to polystyrene (PS) colloids coated with Pluronic™ F108, which has the highest molecular weight and longest PEO chains among the studied block copolymers, prompted a detailed examination of the interaction of such surfactant-treated particulate surfaces with other proteins. Exposure of the coated particles to whole plasma gives rise to a certain polymer displacement with concomitant uptake of a limited amount of protein. This uptake does not result in aggregation nor does it add to the size of each particle. Rather, as shown in a companion study, the repulsion layer established by the stable F108 coating appears to be correlated with the prolonged blood circulation of such coated PS colloids.