Protein adsorption can be reversibly switched on and off on mixed PEO/PAA brushes.

Adsorption of proteins on surfaces placed in biological fluids is a ubiquitous and mostly irreversible phenomenon, desirable or not, but often uncontrolled. Adsorption of most proteins on poly(ethylene oxide) (PEO) brushes is very limited, while the amount of proteins adsorbed on poly(acrylic acid) (PAA) brushes varies with the pH and ionic strength (I) of the protein solution. Mixed brushes of PEO and PAA are designed here to reversibly adsorb and desorb albumin, lysozyme, collagen and immunoglobulin G, four very different proteins in terms of size, solubility and isoelectric point. Protein adsorption and desorption are monitored using X-ray photoelectron spectroscopy, as well as with quartz crystal microbalance for in situ and real-time measurements. Large amounts of protein are adsorbed and then nearly completely desorbed on mixed PEO/PAA brushes by a simple pH and I trigger. The mixed brushes thus nicely combine the properties of pure PAA and pure PEO brushes. These adsorption/desorption cycles are shown to be repeated with high efficiency. The high-performance smart substrates created here could find applications in domains as diverse as biosensors, drug delivery and nanotransport.

Quartz crystal microbalance study of ionic strength and pH-dependent polymer conformation and protein adsorption/desorption on PAA, PEO, and mixed PEO/PAA brushes.

The conformation of polymer chains grafted on a substrate influences protein adsorption. In a previous study, adsorption/desorption of albumin was demonstrated on mixed poly(ethylene oxide) (PEO)/poly(acrylic acid) (PAA) brushes, triggered by solutions of adequate pH and ionic strength (I). In the present work, homolayers of PEO or PAA are submitted to saline solutions with pH from 3 to 9 and I from 10(-5) to 10(-1) M, and their conformation is evaluated in real time using quartz crystal microbalance with dissipation monitoring (QCM-D). Shrinkage/swelling of PAA chains and hydration and salt condensation in the brush are evidenced. The adsorption of human serum albumin (HSA) onto such brushes is also monitored in these different saline solutions, leading to a deep understanding of the influence of polymer chain conformation, modulated by pH and I, on protein adsorption. A detailed model of the conformation of PEO/PAA mixed brushes depending on pH and I is then proposed, providing a rationale for the identification of conditions for the successive adsorption and desorption of proteins on such mixed brushes. The adsorption/desorption of albumin on PEO/PAA is demonstrated using QCM-D.

Adsorption of a PEO-PPO-PEO triblock copolymer on metal oxide surfaces with a view to reducing protein adsorption and further biofouling.

Abstract Biomolecule adsorption is the first stage of biofouling. The aim of this work was to reduce the adsorption of proteins on stainless steel (SS) and titanium surfaces by modifying them with a poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblock copolymer. Anchoring of the central PPO block of the copolymer is known to be favoured by hydrophobic interaction with the substratum. Therefore, the surfaces of metal oxides were first modified by self-assembly of octadecylphosphonic acid. PEO-PPO-PEO preadsorbed on the hydrophobized surfaces of titanium or SS was shown to prevent the adsorption of bovine serum albumin (BSA), fibrinogen and cytochrome C, as monitored by quartz crystal microbalance (QCM). Moreover, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry were used to characterize the surfaces of the SS and titanium after competitive adsorption of PEO-PPO-PEO and BSA. The results show that the adsorption of BSA is well prevented on hydrophobized surfaces, in contrast to the surfaces of native metal oxides.

Design of mixed PEO/PAA brushes with switchable properties toward protein adsorption.

Adsorption of proteins at interfaces is an ubiquitous phenomenon of prime importance. Layers of poly(ethylene oxide) (PEO) are widely used to repel proteins. Conversely, proteins were shown to adsorb deeply into brushes of poly(acrylic acid) (PAA), and their subsequent partial release could be triggered by a change of pH and/or ionic strength (I). Mixed brushes of these polymers are thus promising candidates to tune protein adsorption onto new smart surfaces. In this work, the synthesis of such mixed brushes was performed based on a "grafting to" approach, the two polymers being either grafted sequentially or simultaneously. Detailed characterization of the obtained brushes using static water contact angle measurements, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and polarization-modulation reflection-absorption infrared spectroscopy is presented. While sequential grafting of the two polymers for different reactions times did not give rise to a broad range of composition of mixed brushes, simultaneous grafting of the polymers from solutions with different compositions allows for the synthesis of a range of mixed brushes (mass fraction of PEO in the mixed brushes from 0.35 to 0.65). A key example is then chosen to illustrate the switchable behavior of a selected mixed PEO/PAA brush toward albumin adsorption. The adsorption behavior was monitored with a quartz crystal microbalance. The mixed brush could adsorb high amounts of albumin, but 86% of the adsorbed protein could then be desorbed upon pH and I change. The obtained properties are thus a combination of the ones of PEO and PAA, and a highly switchable behavior is observed toward protein adsorption.

Oligo(ethylene glycol) monolayers by silanization of silicon wafers: Real nature and stability.

Grafting silicon wafers with CH(3)O(CH(2)CH(2)O)(n)C(3)H(6)-trimethoxysilane and -trichlorosilane (n=6 to 9) was performed in different conditions (solvent, reaction time, washing) in order to select procedures compatible with the design of nanostructured surfaces for biomaterial applications, using electron-beam lithography. After a first screening by principal component analysis (PCA), the X-ray photoelectron spectroscopy (XPS) data were analyzed by plotting the carbon to oxygen molar ratio vs the molar ratio of carbon singly bound to oxygen [CO] over carbon bound only to carbon and hydrogen [C(C,H)]. This was found to be a convenient method for discarding samples containing free polymerized silane. Such excess occurred as a result of insufficient washing or unsuitable solvent for the reaction (ether), as confirmed by AFM and thickness measured by X-ray reflectometry. Angle resolved XPS analysis indicated that the grafted silane layer had a 1-2 nm thickness and was covered by a thin layer of adventitious contaminant. As a result, the surface chemical composition obtained covered a broad range (O/C of 0.4 to 1.1; CO/C(C,H) of 2.5 to 6.5); variations could not be related to the nature of the silane reagent and no significant difference was found between hexane and toluene as solvent for the reaction. The grafted silane layer was not stable upon incubation during 24 h in phosphate buffered saline (PBS) at 37 degrees C, which mimics biological environments. As a consequence, the grafted wafers did not show protein repellent properties. This alteration was not observed at room temperature. XPS analysis demonstrated that silane layer detachment was due to a hydrolysis within the SiO(2) layer initially present at the wafer surface.

Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

Adhesion of mammalian cells to polymer surfaces: from physical chemistry of surfaces to selective adhesion on defined patterns.

The study of the adsorption of type I collagen from a solution containing Pluronic F68 has shown that the latter prevents collagen adsorption on polystyrene and does not prevent it on surface-oxidized polystyrene. This explains the control of mammalian cell adhesion by substrate surface hydrophobicity and composition of pre-conditioning solution. On that basis, selective adhesion of different types of mammalian cells (PC12 pheochromocytoma, MSC80 schwannoma, Hep G2 hepatoblastoma, rat hepatocytes) on patterned surfaces was achieved. Therefore tracks (width in the range of a few tens of microm) of reduced hydrophobicity were produced on polystyrene by photolithography and oxygen plasma treatment. After conditioning by a solution containing both Pluronic F68 and extracellular matrix protein (collagen, fibronectin), the latter adsorbed selectively on these paths thus allowing selective adhesion of the cells.