Vladimir Hlady

Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

Protein adsorption on solid surfaces

The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.

From 3D to 2D: A Review of the Molecular Imprinting of Proteins

Molecular imprinting is a generic technology that allows for the introduction of sites of specific molecular affinity into otherwise homogeneous polymeric matrices. Commonly this technique has been shown to be effective when targeting small molecules of molecular weight <1500, while extending the technique to larger molecules such as proteins has proven difficult. A number of key inherent problems in protein imprinting have been identified, including permanent entrapment, poor mass transfer, denaturation, and heterogeneity in binding pocket affinity, which have been addressed using a variety of approaches. This review focuses on protein imprinting in its various forms, ranging from conventional bulk techniques to novel thin film and monolayer surface imprinting approaches.

Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces

Many research and commercial applications use a synthetic substrate which is seeded with cells in a serum-containing medium. The surface properties of the material influence the composition of the adsorbed protein layer, which subsequently regulates a variety of cell behaviors such as attachment, spreading, proliferation, migration, and differentiation. In this study, we examined the relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for MC3T3-E1 osteoblasts on glass surfaces modified with -SO(x), -NH(2), -N(+)(CH(3))(3), -SH, and -CH(3) terminal silanes. We also studied the relationship between cell spread area and migration rate for a variety of anchorage-dependent cell types on a model polymeric biomaterial, poly(acrylonitrile-vinylchloride). Our results indicated that MC3T3-E1 osteoblast behavior was surface chemistry dependent, and varied with individual functional groups rather than general surface properties such as wettability. In addition, cell migration rate was inversely related to cell spread area for MC3T3-E1 osteoblasts on a variety of silane-modified surfaces as well as for different anchorage-dependent cell types on a model polymeric biomaterial. Furthermore, the data revealed significant differences in migration rate among different cell types on a common polymeric substrate, suggesting that cell type-specific differences must be considered when using, selecting, or designing a substrate for research and therapeutic applications.

Adsorption of human serum albumin on precipitated hydroxyapatite

Abstract The adsorption of human serum albumin on precipitated stoichiometric hydroxyapatite Ca10(PO4)6(OH)2, /CaHA/ was followed in a wide pH range. Adsorption was irreversible toward dilution; the isotherms were not of the Langmuir type, but of a stepwise nature. The amount of HSA adsorbed was found to be dependent on the pH and the ionic strength. It decreased with increasing pH, thus indicating the effect of charge repulsion. This effect could be counteracted by increasing the ionic strength, i.e., the concentration of the neutral electrolyte, potassium nitrate. Partial desorption could be effected by increasing the pH of the solution. Adsorption of human serum albumin decreased with increasing temperature.

Fluorescence of adsorbed protein layers. I. Quantitation of total internal reflection fluorescence

Abstract A quantitative total internal reflection intrinsic fluorescence (TIRIF) method for determining the adsorption of proteins at optically suitable solid/liquid interface is presented. Intrinsic protein fluorescence is excited by evanescent part of the standing wave produced by total internal reflection. The TIRIF data are quantified using as an internal standard the fluorescence from nonadsorbed proteins which are present in the evanescent region. In order to account for the fraction of fluorescence excited by scattered light which propagates through and beyond the volume sensed by the evanescent wave, a set of nonadsorbing external standards has to be used. The combination of TIRIF and 125I-protein γ-photon detection system is described and applied to bovine serum albumin (BSA) and human immunoglobulin (IgG) adsorption at silica/electrolyte interfaces. The difference between the results obtained with different adsorption detection systems is discussed. An average fluorescence emission efficiency of adsorbed proteins can be evaluated by combining TIRIF adsorption data with the independent in situ quantitation of protein adsorption. It was found that in some cases adsorbed proteins emit fluorescence with significantly lower quantum yield.

Proteins at Interfaces: Principles, Multivariate Aspects, Protein Resistant Surfaces, and Direct Imaging and Manipulation of Adsorbed Proteins

The principles of protein adsorption are briefly reviewed with emphasis on model proteins at model interfaces. Using a data set for protein behavior at the air/water interface, a multi-variate, multi-axes treatment of protein adsorption behavior is developed: the Tatra plot. By the careful placement and scaling of radial axes, representing 12 protein, surface, and interface parameters, one can begin to deduce various correlations between these parameters. The correlations are then used to formulate hypotheses with which to design additional experiments. The treatment is extended to the solid/liquid interface using data available in the literature for model proteins on model solid surfaces. We then present brief discussion of the extension of the technique to more complex surfaces, including means to parameterize solid/liquid interfacial properties, before proceeding to more complex proteins based on a structural domain approach to protein structure and function. A preliminary analysis employing albumin is briefly presented. We move on to protein resistant surfaces based on polyethylene oxide and present a rationale for the properties and behavior of such interfaces, including a preliminary theoretical model which may be useful for the design and optimization of protein resistant surfaces. Finally, we briefly present some preliminary atomic force microscopy studies of immunoglobulins on mica surfaces, demonstrating not only direct imaging of proteins at interfaces in an aqueous environment but, perhaps even more importantly, their manipulation, processing, and ordering.

Methods for Studying Protein Adsorption

Proteins are interfacially active molecules; a statement that is demonstrated easily by the spontaneous accumulation of proteins at interfaces.1–4 Why do proteins show the propensity to adsorb to interfaces and why do they adsorb so tenaciously? For some proteins, the tendency to adsorb is due to the nature of side chains present on the surface of the protein. Protein is an amphoteric polyelectrolyte.5 Its amino acids have different characteristics: some are apolar and like to be buried inside the protein globule, whereas others are polar and charged and are often found on the outside protein surface. A strong, long-ranged electrostatic attraction between a charged adsorbent and oppositely charged amino acid side chains will lead to a significant free energy change favoring the adsorption process. In other cases, the interfacial activity of the protein may be driven by its marginal structural stability.6 The compactness of the native structure of the protein is due to the optimal amount of apolar amino acid residues. The stability of such a structure depends on the combination of hydrophobic interactions between the hydrophobic side chains, hydrogen bonds between the neighboring side chains and along the polypeptide chains, and the Coulomb interactions between charged residues and van der Waals interactions. An adsorbent surface can “compete” for the same interactions and minimize the total free energy of the system by unfolding the protein structure: the adsorption process may result in a surface-induced protein denaturation.7,8 Elements of the secondary structure of the protein (α helix and β sheet) together with the supersecondary motifs form a compact globular domain. Some proteins are built from more than one domain. In a multidomain protein, it is possible that one domain will dominate the adsorption property of the whole macromolecule at a particular type of interface. For example, acid-pretreated antibodies bind with their constant fragments to a hydrophobic surface.9 In order to completely characterize and predict protein adsorption, one would like to have a quantitative description of adsorption. This description is typically obtained by measuring the adsorption isotherm, adsorption and desorption kinetics, conformation of adsorbed proteins, number and character of protein segments in contact with the surface, and other physical parameters related to the adsorbed protein layer, such as layer thickness and refractive index. This article describes a selected set of techniques and protocols that will provide answers about the mechanism of protein adsorption onto and desorption from surfaces. The reader is referred to the specialized monographs1–4 and a review 10 on protein adsorption for a more comprehensive coverage of various aspects of protein–surface interactions.

Effects of plasma protein adsorption on protein conformation and activity

Protein adsorption at solid-liquid interfaces may lead to significant changes in conformation. Such effects can be monitored in situ for native, unlabelled proteins using the total internal reflection spectroscopy method, by monitoring the UV fluorescence of tryptophan side chains at 320-350 nm. Such studies suggest a partial denaturation of human plasma fibronectin on hydrophobic silica and a blue shift for bovine albumin on hydrophilic silica.

The surface density gradient of grafted poly (ethylene glycol): preparation, characterization and protein adsorption.

A surface density gradient of grafted poly (ethylene glycol) (PEG) chains was prepared using two-phase silanization of a flat silica surface. The first step was to create the surface density gradient of isocyanatopropyldimethylsilyl groups and to hydrolyze the isocyanato moiety into an amine. These surface amines were reacted with an excess of aldehyde-terminated PEG. The PEG-silica surface was characterized by dynamic contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. The length of the PEG gradient region was approximately 7 mm and the thickness in air ranged from zero to 1.1 nm. The maximum surface density of the PEG layer, as calculated from ellipsometric data, amounted to an average 0.4 PEG (molecular weight Mw = 2000 Da) molecule nm-2, while the surface density average of the amine groups was 1.4 molecules nm-2, indicating that only a fraction of the surface amines reacted with aldehyde-terminated PEG. The PEG segment density profile in the gradient PEG region was computed by a self-consistent mean field theory. The PEG (Mw = 2000 Da) segments profile was not parabolic, but showed a thin depletion zone next to the surface. The influence of the surface density of the grafted PEG chains on protein repellence was tested by the adsorption of fibrinogen from solution and from a ternary protein solution mixture containing fibrinogen, albumin and immunoglobulin G. Fibrinogen adsorption onto the silica end of the gradient was extremely low, both in the presence of the other two proteins and in their absence. As the surface density of the grafted PEG chains increased, so did the fibrinogen adsorption (up to 0.024 μg cm-2). It is not clear whether this low fibrinogen adsorption resulted from the interactions of the protein with the grafted PEG chains or with residual surface amines that were available due to some imperfections in the grafted PEG layer.