Enzyme-assisted mineralization of calcium phosphate: exploring confinement for the design of highly crystalline nano-objects.

In hard tissues of vertebrates, calcium phosphate (CaP) biomineralization is a fascinating process that combines specific physicochemical and biochemical reactions, resulting in the formation of extracellular matrices with elegant nanoarchitectures. Although several "biomimetic" strategies have been developed for the design of mineralized nanostructured biointerfaces, the control of the crystallization process remains complex. Herein, we report an innovative approach to overcome this challenge by generating, in situ, CaP precursors in a confined medium. For this purpose, we explore a combination of (i) the layer-by-layer assembly, (ii) the template-based method and (iii) the heterogeneous enzymatic catalysis. We show the possibility of embedding active alkaline phosphatase in a nanostructured multilayered film and inducing the nucleation and growth of CaP compounds under different conditions. Importantly, we demonstrate that the modulation of the crystal phase from spheroid-shaped amorphous CaP to crystalline platelet-shaped hydroxyapatite depends on the degree of confinement of active enzymes. This leads to the synthesis of highly anisotropic mineralized nanostructures that are mechanically stable and with controlled dimensions, composition and crystal phase. The present study provides a straightforward, yet powerful, way to design anisotropic nanostructured materials, including a self-supported framework, which may be used in broad biomedical applications.

Protein-polyelectrolyte complexes to improve the biological activity of proteins in layer-by-layer assemblies.

A standard method of protein immobilization is proposed, based on the use of protein-polyelectrolyte complexes (PPCs) as building blocks for layer-by-layer assembly. Thicker multilayers, with a higher polyelectrolyte fraction, are obtained with PPCs compared to single protein molecules. Biological activity is not only maintained, but specific activity is also higher, as demonstrated for lysozyme-poly(styrene sulfonate) complexes. This is attributed to the more hydrated state of the assemblies. This new method of protein immobilization opens up perspectives for biotechnology and biomedical applications.

Dual stimuli-responsive coating designed through layer-by-layer assembly of PAA-b-PNIPAM block copolymers for the control of protein adsorption.

In this paper, we describe the successful construction, characteristics and interaction with proteins of stimuli-responsive thin nanostructured films prepared by layer-by-layer (LbL) sequential assembly of PNIPAM-containing polyelectrolytes and PAH. PAA-b-PNIPAM block copolymers were synthesized in order to benefit from (i) the ionizable properties of PAA, to be involved in the LbL assembly, and (ii) the sensitivity of PNIPAM to temperature stimulus. The impact of parameters related to the structure and size of the macromolecules (their molecular weight and the relative degree of polymerization of PAA and PNIPAM), and the interaction with proteins under physico-chemical stimuli, such as pH and temperature, are carefully investigated. The incorporation of PAA-b-PNIPAM into multilayered films is shown to be successful whatever the block copolymer used, resulting in slightly thicker films than the corresponding (PAA/PAH)n film. Importantly, the protein adsorption studies demonstrate that it is possible to alter the adsorption behavior of proteins on (PAA-b-PNIPAM/PAH)n surfaces by varying the temperature and/or the pH of the medium, which seems to be intimately related to two key factors: (i) the ability of PNIPAM units to undergo conformational changes and (ii) the structural changes of the film made of weak polyelectrolytes. The simplicity of construction of these PNIPAM block copolymer-based LbL coatings on a large range of substrates, combined with their highly tunable features, make them ideal candidates to be employed for various biomedical applications requiring the control of protein adsorption.

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.

Growth mechanism of confined polyelectrolyte multilayers in nanoporous templates.

We investigate the mechanism of polyelectrolyte multilayer (PEM) assembly in nanoporous templates with a view to synthesizing nanotubes or nanowires under optimal conditions. For this purpose, we focus on the effect of parameters related to the geometrical constraints (pore diameter), the size of the macromolecules (their molar mass and the ionic strength), and the interaction between the pore walls and the adsorbed chains (modulated by the ionic strength). Our results reveal the existence of two regimes in the mechanism of PEM growth: (i) the first regime is comparable to that observed on flat substrates, including the influence of ionic strength and (ii) the second regime, which is slower in terms of kinetics, results from the interconnection established between polyelectrolyte chains across the pores and leads to the formation of a dense gel. As a consequence, the diffusion of polyelectrolytes in nanopores becomes the controlling factor of PEM growth in this second regime. The dense gel, owing to its peculiar structure, enhances the formation of nanowires or of partially occluded nanotubes in some cases, depending on initial pore dimensions.

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.

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy.

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.

Supramolecular assemblies of adsorbed collagen affect the adhesion of endothelial cells.

The behavior of endothelial cells (HUVECs) in contact with thin collagen films presenting different supramolecular organizations was investigated. Collagen was adsorbed on polystyrene (PS) and plasma-oxidized PS (PSox) in conditions ensuring the formation of continuous layers presenting an increasing density of fibrillar structures. Discontinuous collagen layers were also prepared on PS by adsorption followed by dewetting. The morphology of the obtained collagen films was checked by using atomic force microscopy. HUVECs adhesion was evaluated in terms of cell number, cell area, cell shape, and actin structure after 4 h of contact with the prepared collagen layers. In the presence of serum, no adhesion was observed on PS, whereas a substantial adhesion was found on PSox. This is explained by the competition for adsorption, which turns in favor of adhesive proteins secreted by the cells on the hydrophilic PSox, but turns in favor of serum albumin on the hydrophobic PS. The progressive coating of PS by smooth collagen films increased cell adhesion and spreading. However, cell spreading and cytoskeleton organization were adversely affected by the appearance of a high density of collagen fibrillar structures. This latter trend was similarly observed on PSox. On the other hand, HUVECs spreading and cytoskeleton organization were clearly enhanced on discontinuous collagen layers compared with continuous ones. A possible explanation for these observations lies in the modification of exposure and/or spatial distribution of recognition sequences due to spontaneous collagen self-assembly on fibril formation or to collagen aggregation on dewetting.

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.