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.

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.

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.

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.

Functionalization of magnetic nanowires by charged biopolymers.

We report on a facile method for the preparation of biocompatible and bioactive magnetic nanowires. The method consists of the direct deposition of polysaccharides by layer-by-layer (LbL) assembly onto a brush of metallic nanowires obtained by electrodeposition of the metal within the nanopores of an alumina template supported on a silicon wafer. Carboxymethylpullulan (CMP) and chitosan (CHI) multilayers were grown on brushes of Ni nanowires; subsequent grafting of an enzyme was performed by conjugating free amine side groups of chitosan with carboxylic groups of the enzyme. The nanowires are finally released by a gentle ultrasonic treatment. Transmission electron microscopy, electron energy-dispersive loss spectroscopy, and x-ray photoelectron spectroscopy indicate the formation of an homogeneous coating onto the nickel nanowires when one, two, or three CMP/CHI bilayers are deposited. This easy and efficient route to the biochemical functionalization of magnetic nanowires could find widespread use for the preparation of a broad range of nanowires with tailored surface properties.

Mechanical properties of nanotubes of polyelectrolyte multilayers.

The elastic properties of nanotubes fabricated by layer-by-layer (LbL) assembly of polyelectrolytes in the nanopores of polycarbonate track-etched membranes have been investigated by resonant contact Atomic Force Microscopy (AFM), for nanotube diameters in the range of 100 to 200 nm. The elastic modulus of the nanotubes was computed from the resonance frequencies of a cantilever resting on freely suspended LbL nanotubes. An average value of 115 MPa was found in air for Young's modulus of these nanostructures, well below the values reported for dry, flat multilayers, but in the range of values reported for water-swollen flat multilayers. These low values are most probably due to the lower degree of ionic cross-linking of LbL nanotubes and their consequently higher water content in air, resulting from the peculiar mode of growth of nanoconfined polyelectrolyte multilayers.

Electrochemical synthesis of conjugated polymer wires and nanotubules

Two electrochemical methods designed for the synthesis of polypyrrole (PPy) wires and nanotubules are the topic of this paper. The concept that allows the morphology of PPy to be controlled is discussed for each method, and the performances of the two complementary techniques are compared in terms of dimension, shape and conductivity of PPy.

Elastic modulus of polypyrrole nanotubes

The first measurements of the tensile elastic modulus of polypyrrole nanotubes are presented. The nanotubes were mechanically tested in three points bending using atomic force microscopy. The elastic tensile modulus was deduced from force-curve measurements on different nanotubes with outer diameter ranging between 35 and 160 nm. It is shown that the elastic modulus strongly increases when the thickness or outer diameter of polypyrrole nanotubes decreases.