Molecular simulations were performed to study the interactions between a protein (lysozyme, LYZ) and phosphorylcholine-terminated self-assembled monolayers (PC-SAMs) in the presence of explicit water molecules and ions. The results show that the water molecules above the PC-SAM surface create a strong repulsive force on the protein as it approaches the surface. The structural and dynamic properties of the water molecules above the PC-SAM surface were analyzed to provide information regarding the role of hydration in surface resistance to protein adsorption. It can be seen from residence time dynamics that the water molecules immediately above the PC-SAM surface are significantly slowed down as compared to bulk water, suggesting that the PC-SAM surface generates a tightly bound, structured water layer around its head groups. Moreover, the orientational distribution and reorientational dynamics of the interfacial water molecules near the PC-SAM surface were found to have the ionic solvation nature of the PC head groups. These properties were also compared to those obtained previously for an oligo(ethylene glycol) (OEG) SAM system and bulk water.

A significant challenge in the field of biomaterials is the nonspecific adsorption of proteins. It has been suggested that overall neutral materials composed of mixed charge components present protein-resistant properties. This work describes the development of a novel nonfouling polymer brush formed from a surfaceinitiated, two-component atom transfer radical polymerization. The polymer brushes are composed of varying mixtures of positively and negatively charged monomers depending on the polymerization conditions. The polymer brushes were characterized by both atomic force microscopy and electron spectroscopy for chemical analysis to determine the thickness and composition, respectively. The nonspecific adsorption of fibrinogen, lysozyme, and bovine serum albumin was measured using a surface plasmon resonance biosensor. It was found that when the polymer brush surface coating was formed as a statistical copolymer from the two charged components, it had nonfouling properties for all three probe proteins.

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Interpenetrating polymer networks (IPNs) were prepared by the modification of a segmented polyurethane (SPU) with a cross-linked sulfobetaine methacrylate (SBMA) polymer. The IPN films that were prepared can effectively resist nonspecific protein adsorption when the distribution of SBMA units within the SPU film is well controlled, and they retain high mechanical strengths inherent from the base SPU films. Furthermore, the zwitterionic and biomimetic nature of sulfobetaine and the ease of SBMA preparation make SBMA-based materials very attractive for a wide range of applications. It is challenging to control the diffusion of highly polar SBMA into the hydrophobic network of SPU. In this study, various parameters governing the formation of IPNs containing SBMA were studied. The chemical composition depth profile of the IPN films was determined by confocal Raman microscopy. The morphology and thickness of these IPN films were examined by atomic force microscopy and scanning electron microscopy. The amount of adsorbed proteins on the IPN films was determined by an enzyme-linked immunosorbent assay. Results show that the amount of adsorbed proteins on the IPN films depends on the incubation conditions, including solvent polarity, incubation time, SBMA monomer ratio, and incubation concentration. It appears that the IPN films prepared in a mixed solvent of higher polarity with long incubation time lead to very low protein adsorption. This study not only introduces a new IPN system containing SBMA, but also provides a fundamental understanding of various parameters governing the formation of IPNs.

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Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.

The prevention of nonspecific protein adsorption to synthetic materials and devices presents a major design challenge in the biomedical community. While some chemical groups can resist nonspecific protein adsorption from simple solutions for limited contact times, there remains a need for new nonfouling functional groups and surface coatings that prevent protein adsorption from complex media like blood or in harsh environments like seawater. Recent studies of the molecular mechanisms of nonfouling surfaces have identified a strong correlation between surface hydration and resistance to nonspecific protein adsorption. In this work, we describe a simple experimental method for evaluating the intrinsic hydration capacity of model surface coating functional groups based on the partial molal volume at infinite dilution. In order to evaluate a range of hydration capacity and nonfouling performance, solutes were selected from three classes: ethylene glycols, sugar alcohols, and glycine analogues. The number of hydrating water molecules bound to a solute was estimated by comparing the molecular volume at infinite dilution to the solute van der Waals molecular volume. The number of water molecules associated with each solute was further validated by constant pressure and temperature molecular dynamics simulations. Finally, a size-normalized molecular volume was correlated to previously observed protein adsorption experiments to relate the intrinsic hydration capacity of functional groups to their known nonfouling abilities.

Materials that are resistant to nonspecific protein adsorption are critical in the biomedical community. Specifically, nonfouling implantable biomaterials are necessary to reduce the undesirable, but natural foreign body response. The focus of this investigation is to demonstrate that polyampholyte hydrogels prepared with equimolar quantities of positively charged [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged 2-carboxyethyl acrylate (CAA) monomers are a viable solution to this problem. TMA/CAA hydrogels were prepared and their physical and chemical properties were characterized. The fouling resistance of the TMA/CAA hydrogels were assessed at varying cross-linker densities using enzyme-linked immunosorbant assays (ELISAs). The results clearly demonstrate that TMA/CAA hydrogels are resistant to nonspecific protein adsorption. A unique advantage of the fouling resistant TMA/CAA system is that bioactive proteins can be covalently attached to these materials using standard conjugation chemistry. This was demonstrated in this study through a combination of ELISA investigations and short-term cell adhesion assays. The multifunctional properties of the TMA/CAA polyampholyte hydrogels shown in this work clearly demonstrate the potential for these materials for use as tissue regeneration scaffolds for many biomedical applications.