James M. Van Alstine

Grafting Poly(ethylene glycol) Epoxide to Amino-Derivatized Quartz: Effect of Temperature and pH on Grafting Density.

Microparticle capillary electrophoresis was used to characterize the surface of quartz capillaries grafted with the glycidyl ether of poly(ethylene glycol) (E-PEG). Site dissociation modeling of capillary electrokinetic behavior provided estimates of surface group pK and density, plus the distance (d) from the surface to the hydrodynamic plane of shear. Native quartz appeared to possess silanol groups of pK 3.6 and 6.9 whose surface densities varied with quartz treatment. Aminopropylsilane derivatization of quartz silanol groups in toluene yielded a coating which was stable (>6 h) at pH 10.3 and 60 °C. Aqueous grafting of E-PEG to this surface was relatively independent of pH (7.3-10.3) and reaction time (6-24 h) but was significantly influenced by reaction temperature (25-95 °C) and salt composition. PEG-grafted capillaries exhibited greatly reduced electroosmosis from pH 2 to 11. Significant grafting could be obtained under mild conditions (6 h, 35 °C, 0.4 M K(2)SO(4), pH 6.9). These results suggest that PEG chains increasingly extend normal to a surface as their grafting density increases, and that PEG conformation influences grafting density. The methods described should aid the use of PEG-coated surfaces in a variety of applications.

Electrokinetic characterization of hydrophilic polymer coatings of biotechnical significance

Abstract Analytical microparticle electrophoresis was used to characterize various polymer coatings known to control protein adsorption and related phenomena of biotechnical significance. The electrophoretic mobility of polystyrene latex microspheres and the electro-osmosis associated with quartz capillaries were characterized over the pH range 2–11. Such characterization provides information related to surface modification. Aminopropylsilane and mercaptopropylsilane were shown to be effective sublayers for covalent attachment of hydrophilic polymers to quartz glass surfaces. Poly(ethylenimine) was similarly verified as an effective sublayer for polystyrene latex. Polymer coatings based on poly(ethylene glycol) and three polysaccharides, dextran, ethyl(hydroxyethyl)cellulose, and hydroxypropylcellulose, were found to reduce capillary electro-osmosis and microsphere electrophoretic mobility significantly over a broad pH range. This reduction corresponds to the ability of these coatings to reduce protein adsorption and control surface wetting by aqueous polymer two-phase systems.

TRIANGULAR PHASE DIAGRAMS TO PREDICT THE FRACTIONATION OF FREE FATTY ACID MIXTURES VIA UREA COMPLEX FORMATION

The authors previously developed a rapid and inexpensive procedure to fractionate free fatty acid (FFA) mixtures via urea complex UC formation. The process yields two phases in equilibrium, a solvent-rich extract phase containing unsaturated FFA and a solid raffinate phase consisting of UCs of saturated and monounsaturated FFA. It effectively removed saturated FFA, that is, palmitic and stearic acid, from an FFA mixture derived from low erucic acid rapeseed (LEAR) oil. In the present study, triangular phase diagrams were determined for the urea/ethanol/water/LEAR-FFA system at various temperatures, which delineated the phase boundary between the 1-phase and 2-phase regions. Calculations of the amounts and distribution of urea and LEAR-FFA between the two phases based on tie-lines from the phase diagram strongly agreed with experimental data. The diagram accurately predicted the temperature required to cosolubilize urea and LEAR-FFA in solvent. A plot of the percent extraction of an individual FFA species versus percent extraction of total FFA for all major FFA species yielded a universal curve describing results related to various combinations of urea, water, ethanol, and LEAR-FFA. Equations derived from the phase diagram, distribution data for the FFA species, and mass balances were combined to create a mathematical model, which accurately predicted the FFA composition of both phases. The methodology used and results obtained should aid critical evaluation of large scale UC-based FFA fractionation procedures.

Critical micelle concentration dependence on head-group size in polyoxyethylene nonionic surfactants

Abstract The critical micelle concentrations (CMCs) of a number of nonionic surfactants composed of fatty acids esterified to polyoxyethylene molecules containing large numbers of oxyethylene units ( E ) were determined fluorimetrically using 8-anilino-1-naph-thalenesulfonate. These CMCs were found to be two orders of magnitude lower than the values predicted from an extrapolation of log CMC versus E plots derived from literature data on relatively low-molecular-weight homologues. Our results suggest that the CMC values of such surfactants may be better extrapolated from plots of log CMC versus log E .

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants.

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.

The wetting behavior of aqueous two-phase polymer test systems on dextran coated glass surfaces: Effect of molecular weight

Abstract The wetting behavior of phase-separated aqueous solutions of dextran and poly (ethylene glycol) on quartz substrates coated with covalently bound dextran fractions was examined as a function of coating molecular weight. The effect of dissolved polymer size was also investigated by using two-phase test systems containing high and low molecular weight dextrans. Sharply cut dextran fractions of Mw from 27,900 to 205,000 were attached to quartz activated with 3-aminopropyldimethylethoxysilane through reductive amination of the dextrans reducing end group in the presence of sodium cyanoborohydride. X-ray photoelectron spectroscopy and contact angle measurements in the two-phase polymer solutions were used to assess the quantity and quality of the surface coatings. Our results demonstrate that wetting by the dextran-rich phase improves, as evidenced by a declining contact angle, with increasing molecular weight of the bound dextran, decreasing molecular weight of the free dextran in solution and increasing time of exposure to the polymer phases. The usual relationship of increasing contact angle with increasing interfacial tension is not observed in these phase-separated polymer mixtures.

Polymer coatings for improved protein crystal growth

Abstract The ability to grow quality protein crystals is necessary to analyze protein structure by X-ray diffraction and related techniques. As such it plays a key role in enzymology, structure-based drug design, molecular biology, and other biomedical areas. It is also required for macromolecule purification by crystallization. Protein crystal growth (PCG) may be negatively influenced by various factors related to nonspecific adsorption and adherence at growth chamber surfaces. Such factors include nucleation and growth of flawed crystals at chamber walls, or wall growth blockage of optical monitoring paths. Surface localized poly(ethylene glycol) (PEG) and other neutral, hydrophilic polymers are known to significantly reduce nonspecific adsorption of biological macromolecules and particles. Preliminary studies, involving various PCG methods (temperature induction, vapor diffusion), apparatii (test tubes, cuvettes, and specialized PCG hardware), growth chamber materials (glass, polystyrene, polysulfone), chamber volumes (0.1–10 ml) and protein samples (lysozyme, thaumatin, insulin) indicate the potential of PEG coatings to significantly reduce problems related to adsorption in PCG. The results, which match the ability of such coatings to reduce protein adsorption as evaluated by both ellipsometry and enzyme linked immunoassay, are discussed in relation to colloidal stabilization theory and properties of PEG coated surfaces.

Automated Particle Electrophoresis: Modeling and Control of Adverse Chamber Surface Properties

Electrophoretic analysis of colloidal particles is adversely affected by a host of surface phenomena, including electroosmosis, phase wall wetting, and sample or air bubble adsorption. Neutral, hydrophilic polymer coatings control such phenomena on a variety of surfaces. Poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) conjugates significantly reduce electroosmosis and positively control adsorption and wetting in the glass sample chambers (5 mm × 3 mm × 1 mm i.d.) employed in a representative commercial electrophoresis apparatus (Coulter DELSA 440). The reduction in electroosmosis (e.g., 80% in 7.5 mM solution at pH 11) was similar to that exhibited by coated 2-mm-i.d. quartz capillaries in a Rank MK I manual apparatus. PEG-PEI coatings significantly reduce electroosmosis over a wide range of pH (2-11) and ionic strength (1-100 mM) and can be stable for weeks under normal laboratory conditions. They greatly enhance ease of operation and accuracy (sample mean electrophoretic mobility ± SD) of the DELSA 440. The latter results from reduced electroosmosis flow profile gradients near the chamber center-axis stationary levels, where particle mobility is typically measured. Such flow profiles may also be affected by chamber wall surface asymmetries. A hydrodynamic description of electroosmotic fluid flow in rectangular chambers was adapted in order to analyze the propagation of errors due to both nonideal focusing and chamber surface asymmetry. The analysis indicated that the accuracy of rectangular chambered devices may be improved by measuring particle mobility at stationary levels different than chamber center-axes. As a result, some rectangular chambers may confer accuracy advantages over cylindrical chambers.

Poly(ethylene glycol) amphiphile adsorption and liposome partition

Surface localized poly(ethylene glycol) (PEG) amphiphiles of type C16:0-EO151 and C18:2-EO151 were studied via ellipsometry at macroscopic, flat methylated silica (MeSi), phosphatidic acid (PA), and phosphatidylcholine (PC) surfaces. At these surfaces the amphiphiles adsorb similarly, in a non-cooperative manner, achieving a plateau (approximately 0.1 PEG chains/nm2) well below amphiphile critical micelle concentration (CMC). The resultant PEG-enriched layers were 10-15 nm thick, with a polymer concentration (approximately 0.07 g/cm3) greater than the PEG-enriched phase of many dextran, PEG aqueous two-phase systems. PEG-amphiphile adsorption (mg/m2) at hydrophobic and phospholipid flat surfaces correlated with changes in the partition (log K) of PC liposomes in such two-phase systems. PEG-amphiphile adsorption at macroscopic surfaces appears to represent a balance between hydrophobic attraction and repulsive intra-chain interactions which promote chain elongation normal to the surface.