L. Zhang, C. Carucci, S. Reculusa, B. Goudeau, P. Lefrançois, S. Gounel, N. Mano, A. Kuhn
The immobilization of bilirubin oxidase (BOD) on macroporous gold electrodes for the optimization of bioelectrocatalytic activity is described. A bilirubin oxidase mutant S362C (cys‐BOD) engineered with a cysteine residue located on purpose at the enzyme surface close to the T1 active center was used. It allows the attachment in one‐step of a self‐assembled monolayer of the enzyme to gold through a reaction between the thiol group of the cysteine residue and the metal surface. BOD immobilization of wild type and S362C mutant in macroporous gold electrodes allowed high retention of activity and perfect control of the overall BOD loading due to the fine‐tuning of the macroporous structure. The macroporous arrangement together with the use of cys‐BOD makes these rationally designed enzyme‐modified electrodes very promising candidates for high‐performance bioelectrocatalytic devices with improved activity and stability.
A. Perro, L. Giraud, N. Coudon, S. Shanmugathasan, B. Goudeau, J.-P. Douliez and V. Ravaine
Coacervation is a phase separation process involving two aqueous phases, one solute-phase and one solute-poor phase. It is frequently observed among oppositely-charged polyelectrolyte systems. In this study, we focus on self-coacervation involving a single polymer chain and investigate its potential for encapsulation applications. Negatively charged polyacrylic acid polymer chains were partially cationized using diamine and carbodiimide chemistry affording ampholytes, named PAA-DA, with tunable charge ratio. When dispersed in water, at pH 7, PAA-DA was soluble but a phase separation occurs when decreasing pH close to the isoelectric point. Coacervation is found only for a given amine-to-acid ratio otherwise precipitation is observed. Increasing the pH above 4 yielded progressive destruction of the coacervates droplets via the formation of vacuoles within droplets and subsequent full homogeneous redispersion of PAA-DA in water. However, addition of calcium allowed increasing the coacervate droplet stability upon increasing the pH to 7 as the divalent ion induced gelation within droplets. Moreover, the coacervate droplets present the ability to spontaneously sequestrate a broad panel of entities, from small molecules to macromolecules or colloids, with different charges, size and hydrophobicity. Thanks to the reversible character of the coacervates, triggered-release could be easily achieved, either by varying the pH or by removing calcium ions in the case of calcium-stabilized coacervates. Self-coacervation presents the advantage of pathway-independent preparation, offering a real output interest in pharmacy, water treatment, food science or diagnostics.
S. Ramalingam, G. Le Bourdon, E. Pouget, A. Scalabre, J. Raghava Rao and A. Perro
The design of nanocarriers containing hydrophobic and hydrophilic compounds represents a powerful tool for cocktail delivery. Water-in-oil-in-water emulsions constitute an attractive approach as they offer dual encapsulation and provide a template for the constitution of a capsule. A limitation in the preparation of nano double emulsions is their instability resulting from high curvature radii. In this work, silica nanocapsules (NCs) stable over several months were synthesized. This was achieved by exploiting a double emulsion in which the oil phase is constituted by a combination of oils presenting several volatilities. The decrease of oil droplets size by evaporation favored the deposition of a silica layer at the nanoscale interface. The release of the payload obtained by drying the capsules was investigated by fluorescence spectroscopy. Understanding the interactions between proteins and nanocapsules is a fundamental point for many biological applications. Nanocapsules were exposed to two model proteins, which were Bovine Serum Albumin (BSA) and Lysozyme (Ly). These proteins, presenting differences in charges and size, showed distinctive arrangements onto the nanocapsules. Moreover, we have studied changes in α-helix and β-sheet content, which divulged the interactions between the proteins and the nanocapsules.
M.-C. Tatry, E. Laurichesse, A. Perro, V. Ravaine and V. Schmitt
The aim of the paper is to examine the adsorption kinetics of soft microgels and to understand the role off undamental parameters such as electrostatics and deformability on the process. This knowledge is further exploited to produce microgel-stabilized emulsions using a co-flow microfluidic device.Uncharged microgels made of poly(N-isopropylacrylamide) are synthesized with variable cross-linker contents, and charged ones are produced by introducing pH sensitive co-monomers during the synthesis.The study is carried out by measuring the microgels adsorption kinetics by means of the pendant drop method. The surface pressure is derived from the previous results as a function of time and is measuredas a function of the area compression using a Langmuir trough. Emulsions are produced using a microfluidicdevice varying the microgels concentration and their stability is visually assessed.The microgels deformability as well as higher particle concentrations favour their adsorption. The adsorption is not governed by diffusion, it is cooperative and irreversible. Conversely, the kinetics is slowed down for increasing cross-linking density. The presence of charges slows down the kinetics of adsorption. In the presence of electrolyte, the kinetics accelerates and becomes similar to the one of neutral microgels. The original features of microgel adsorption is highlighted and the differences with adsorption of polymers, star polymers, proteins, and polyelectrolytes are emphasized. Taking benefit from the adsorption kinetics, the required formulation conditions for producing microgel-stabilized emulsions using a co-flow microfluidic device are derived.There exists a critical concentration above which microgels spontaneously adsorb in a sufficient way to decrease the interfacial tension. This critical microgel concentration increases with the cross-linking density and is higher for charged microgels. Whatever the kinetics, the same surface pressure is finally reached. This peculiar behaviour is likely a consequence of the presence of dangling chains in the as-prepared microgels. Consequently, a microgel excess is required to produce emulsions using microfluidics where adsorption has to be spontaneous.
H. Labie, A. Perro, V. Lapeyre, B. Goudeau, B. Catargi, R. Auzély, V. Ravaine
A simple route to deliver on demand hydrosoluble molecules such as peptides, packaged in biocompatible and biodegradable microgels, is presented. Hyaluronic acid hydrogel particles with a controlled structure are prepared using a microfluidic approach. Their porosity and their rigidity can be tuned by changing the crosslinking density. These negatively-charged polyelectrolytes interact strongly with positively charged linear peptides such as poly-L-lysine (PLL). Their interactions induce microgel deswelling and inhibit microgel enzymatic degradability by hyaluronidase. While small PLL penetrate the whole volume of themicrogel, PLL larger than the mesh size of the network remain confined at its periphery. They make a complexed layer with reduced pore size, which insulates the microgel inner core from the outer medium. Consequently, enzymatic degradation of the matrix is fully inhibited and non-affinity hydrophilic species can be trapped in the core. Indeed, negatively charged or small neutral peptides, without interactions with the network,usually diffuse freely across the network. By simple addition of large PLL, they are packaged in the core and can be released on demand, upon introduction of an enzyme that degrades selectively the capping agent. Single polyelectrolyte layer appears as a simple generic method to coat hydrogel-based materials of various scales for encapsulation and controlled delivery of hydrosoluble molecules.
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