Enzymatic cascade reaction in simple-coacervates.

The design of enzymatic droplet-sized reactors constitutes an important challenge with many potential applications such as medical diagnostics, water purification, bioengineering, or food industry. Coacervates, which are all-aqueous droplets, afford a simple model for the investigation of enzymatic cascade reaction since the reactions occur in all-aqueous media, which preserve the enzymes integrity. However, the question relative to how the sequestration and the proximity of enzymes within the coacervates might affect their activity remains open. Herein, we report the construction of enzymatic reactors exploiting the simple coacervation of ampholyte polymer chains, stabilized with agar. We demonstrate that these coacervates have the ability to sequester enzymes such as glucose oxidase and catalase and preserve their catalytic activity. The study is carried out by analyzing the color variation induced by the reduction of resazurin. Usually, phenoxazine molecules acting as electron acceptors are used to characterize glucose oxidase activity. Resazurin (pink) undergoes a first reduction to resorufin (salmon) and then to dihydroresorufin (transparent) in presence of glucose oxidase and glucose. We have observed that resorufin is partially regenerated in the presence of catalase, which demonstrates the enzymatic cascade reaction. Studying this enzymatic cascade reaction within coacervates as reactors provide new insights into the role of the proximity, confinement towards enzymatic activity.

Responsive microgels-based colloidosomes constructed from all-aqueous pH-switchable coacervate droplets.

HYPOTHESIS: Colloidosomes made of stimuli-responsive microgels offer the opportunity to design polymeric capsules with a hierarchical and tunable pore distribution. Coacervates stabilized by a microgel monolayer represent a unique strategy to build colloidosomes from all-aqueous emulsion drops, while exploiting the sequestration and dissolution properties of the coacervates. EXPERIMENTS: Methacrylated poly(N-isopropylacrylamide) (pNIPAM) microgels are used to stabilize coacervates made of an ampholyte polymer at a pH close to its isoelectric point. They are further cross-linked under UV-irradiation. The resulting assemblies are studied by means of confocal microscopy. Their permeability towards dextrans and nanoparticles is studied before and after dissolution of the coacervate. FINDINGS: PNIPAM microgels are found to stabilize the coacervates by adsorbing at their surface. Inter cross-linking the microgels results in the formation of an elastic colloidosome that persists after the coacervate dissolution and withstands surface deformations up to about 200%. The coacervate is exploited as a sequestrating core to entrap a water-soluble payload, which can be further released upon coacervate dissolution, while the membrane exhibits a size-selecting permeability. The membrane properties can also be switched by the volume phase transition of the microgels. Coacervate-embedded colloidosomes open new perspectives in the area of encapsulation/extraction and controlled transport of water-soluble/dispersed species.

Building micro-capsules using water-in-water emulsion droplets as templates.

The use of templates in materials chemistry is a well-established approach for producing membrane-bounded hollow spheres used for microencapsulation applications, but also in synthetic biology to assemble artificial cell-like compartments. Sacrificial solid or gel micro-particles, but also liquid-like oil-in-water or water-in-oil emulsion droplets are routinely used as templates to produce capsules. Yet, disruption of the core sacrificial material often requires harsh experimental conditions, such as organic solvents, which limits the use of such approach to encapsulate fragile solutes, including biomolecules. Recently, water-in-water emulsion droplets have emerged as promising alternative templates to produce capsules in solvent-free conditions. These water-in-water droplets result from liquid-liquid phase separation in dilute aqueous polymer or surfactants solutions. Their ease of preparation, the large palette of components they can be assembled from and the lack of harsh solvent or oil used for their production make water-in-water emulsions of practical importance in materials chemistry. Water-in-water droplets can also spontaneously sequester solutes by equilibrium partitioning, which provides a simple strategy to locally accumulate molecules of interest and encapsulate them in capsules after interfacial shell formation. Here, we review recent works that employ water-in-water emulsion droplets to prepare capsules and suggest possible additional applications in materials chemistry.

Versatile template-directed synthesis of gold nanocages with a predefined number of windows.

Highly symmetrical gold nanocages can be produced with a controllable number of circular windows of either 2, 3, 4, 6 or 12 via an original fabrication route. The synthetic pathway includes three main stages: the synthesis of silica/polystyrene multipod templates, the regioselective seeded growth of a gold shell on the unmasked part of the silica surface and the development of gold nanocages by dissolving/etching the templates. Electron microscopy and tomography provide evidence of the symmetrical features of the as-obtained nanostructures. The optical properties of nanocages with 4 and 12 windows were measured at the single particle level by spatial modulation spectroscopy and correlated with numerical simulations based on finite-element modeling. The new multi-step synthesis approach reported here also allows the synthesis of rattle-like nanostructures through filling of the nanocages with a guest nano-object. With the potential to adjust the chemical composition, size and geometry of both the guest particle and the host cage, it opens new routes towards the fabrication of hollow nanostructures of high interest for a variety of applications including sensing devices, catalytic reactors and biomedicine.

Chiral Macroporous MOF Surfaces for Electroassisted Enantioselective Adsorption and Separation.

The development of surfaces with chiral features is a fascinating challenge for modern materials science, especially when they are used for chiral separation technologies. In this contribution, the design of hierarchically structured chiral macroporous zeolitic imidazolate framework-8 (ZIF-8) electrodes is presented. They are elaborated by an electrochemical deposition-dissolution technique based on the electrodeposition of metal through a colloidal crystal template, followed by controlled electrooxidation. This generates locally metal cations, which can interact with a chiral ligand present in the solution to form metal-organic frameworks (MOFs). The macroporous structure facilitates the access of the chiral recognition sites, located in the mesoporous MOF, and thus helps to overcome mass transport limitations. The efficiency of the designed functional materials for chiral adsorption and separation can be fine-tuned by applying an adjustable electric potential to the electrode surfaces. This hierarchical chiral ZIF-8 structure was deposited at the walls of a microfluidic device and used as a stationary phase for enantioselective separation. The potential-controlled interaction between the stationary phase and the chiral analytes allows baseline separation of two enantiomers. This opens up interesting perspectives for using hierarchically structured chiral MOFs as an efficient material for the selective adsorption and separation of chiral compounds.

Stabilization of All-in-Water Emulsions To Form Capsules as Artificial Cells.

Building artificial cells through a bottom-up approach is a remarkable challenge that would be of interest for our understanding of the origin of life, research into the minimal conditions required for life, the formation of bioreactors, and for industrial applications. To date, capsules such as liposomes, including polymersomes, are widely used, but the low membrane permeability and method to encapsulate biological materials within these structures hamper their use. By contrast, all-in-water emulsion droplets, including coacervate droplets, are promising compartments, mainly because they can spontaneously sequester chemicals. However, they lack a membrane necessary to control exchange between the inner and outer media. Moreover, droplets tend to coalesce with time, yielding macroscopic phase separation that is deleterious for any use as artificial cells. Recent advances, which are reviewed herein, have shown that such droplets can be stabilized by using lipid membranes, liposomes, polymers, proteins, and particles, and thus, preventing coalescence. Finally, different strategies that could allow the future development of artificial cells from these stabilized all-in-water emulsion droplets are discussed.

Kinetics of spontaneous microgels adsorption and stabilization of emulsions produced using microfluidics.

The aim of the paper is to examine the adsorption kinetics of soft microgels and to understand the role of fundamental 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 measured as a function of the area compression using a Langmuir trough. Emulsions are produced using a microfluidic device 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.

Self-coacervation of ampholyte polymer chains as an efficient encapsulation strategy.

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.

Adsorption of Proteins on Dual Loaded Silica Nanocapsules.

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 droplet 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 ß-sheet content, which divulged the interactions between the proteins and the nanocapsules.

Sealing hyaluronic acid microgels with oppositely-charged polypeptides: A simple strategy for packaging hydrophilic drugs with on-demand release.

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 the microgel, 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.

Potential-Induced Fine-Tuning of the Enantioaffinity of Chiral Metal Phases.

Concepts leading to single enantiomers of chiral molecules are of crucial importance for many applications, including pharmacology and biotechnology. Recently, mesoporous metal phases encoded with chiral information have been developed. Fine-tuning of the enantioaffinity of such structures by imposing an electric potential is proposed, which can influence the electrostatic interactions between the chiral metal and the target enantiomer. This allows the binding affinity between the chiral metal and the target enantiomer to be increased, and thus, the discrimination between two enantiomers to be improved. The concept is illustrated by generating chiral encoded metals in a microfluidic channel by reduction of a platinum salt in the presence of a liquid crystal and l-tryptophan as a chiral model template. After removal of the template molecules, the modified microchannel retains a pronounced chiral character. The chiral recognition efficiency of the microchannel can be fine-tuned by applying a suitable potential to the metal phase. This enables the separation of both components of a racemate flowing through the channel. The approach constitutes a promising and complementary strategy in the frame of chiral discrimination technologies.

Preparation of Template-Free Robust Yolk-Shell Gelled Particles from Controllably Evolved All-in-Water Emulsions.

A template-free all-aqueous bulk preparation of robust hollow capsules having a gelatin shell from all-in-water double emulsions is reported. The hot (>40 °C) quaternary system water/polyethylene glycol (PEG)/gelatin/alginate is shown to spontaneously form PEG-in-gelatin-in-PEG double water emulsion droplets having a multinuclear core. These droplets are stable upon cooling below the temperature at which gelatin gelled. In contrast, above the melting temperature of gelatin, multinuclear double emulsion droplets controllably evolve into stable mononuclear yolk (aqueous PEG)-shell (gelatin) capsules dispersed in the aqueous PEG continuous phase. It is demonstrated that the gelatin shell can accommodate negatively charged latex beads and be re-enforced by glutaraldehyde or silica. These capsules are also shown to encapsulate payloads, suggesting possible applications in microencapsulation, drug delivery, and synthetic biology.

Core-shell colloidal particles with dynamically tunable scattering properties.

We design polystyrene-poly(N'-isopropylacrylamide-co-acrylic acid) core-shell particles that exhibit dynamically tunable scattering. We show that under normal solvent conditions the shell is nearly index-matched to pure water, and the particle scattering is dominated by Rayleigh scattering from the core. As the temperature or salt concentration increases, both the scattering cross-section and the forward scattering increase, characteristic of Mie scatterers. The magnitude of the change in the scattering cross-section and scattering anisotropy can be controlled through the solvent conditions and the size of the core. Such particles may find use as optical switches or optical filters with tunable opacity.

Synthesis of multivalent silica nanoparticles combining both enthalpic and entropic patchiness.

Silica particles with a controlled number of entropic patches, i.e. dimples, are synthesized through the growth of the silica core of binary multipods that have been produced by a seeded-growth emulsion polymerization reaction. Transmission electron microscopy studies indicate that the silica surface conforms to the shape of the polystyrene (PS) nodules of the multipods while growing, allowing good control of the final shape of the dimpled silica particles. The PS nodules are also used as protecting masks to regioselectively graft amino groups, as revealed by the adsorption of gold markers. After dissolution of the PS nodules, some polymer chains remain grafted onto the silica surface, forming organic bumps. These residues are also selectively functionalized, leading to silica particles with both entropic and enthalpic patches.

Design and elaboration of colloidal molecules: an overview.

The concept of colloidal molecules was first evoked by van Blaaderen in 2003 for describing small non-spherical colloids made of the aggregation of a small number of particles. He predicted original properties to the complex assemblies of such colloids, in particular in optics. This critical review deals with the different strategies reported for creating robust clusters of spherical particles which could mimic the space-filling models of simple conventional molecules. These routes concern either the controlled clustering of preformed colloids directed by coalescence, physical routes, chemical routes, or 2-D/3-D geometrical confinement, or strategies starting from a single colloid which is decorated by satellite colloids by taking advantage of controlled phase separation or nucleation and growth phenomena. These routes are compared from the viewpoint of the accessible shapes, their tunability and scalability (146 references).

Mastering a double emulsion in a simple co-flow microfluidic to generate complex polymersomes.

We show that the production and the geometrical shape of complex polymersomes can be predicted by varying the flow rates of a simple microdevice using an empirical law which predicts the droplet size. This device is constituted of fused silica capillaries associated with adjusted tubing sleeves and T-junctions. Studying the effect of several experimental parameters, double emulsions containing a controlled number of droplets were fabricated. First, this study examines the stability of a jet in a simple confined microfluidic system, probing the conditions required for droplets production. Then, multicompartmental polymersomes were formed, controlling flow velocities. In this work, poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) and poly(butadiene)-block-poly(ethyleneoxide) (PBut-b-PEO) amphiphilic copolymers were used and dissolved in chloroform/cyclohexane mixture. The ratio of these two solvents was adjusted in order to stabilize the double emulsion formation. The aqueous suspension contained poly(vinyl alcohol) (PVA), limiting the coalescence of the droplets. This work constitutes major progress in the control of double emulsion formation in microfluidic devices and shows that complex structures can be obtained using such a process.

Bulk synthesis of polymer-inorganic colloidal clusters.

We describe a procedure to synthesize colloidal clusters with polyhedral morphologies in high yield (liter quantities at up to 70% purity) using a combination of emulsion polymerization and inorganic surface chemistry. We show that the synthesis initially used for silica-polystyrene hybrid clusters can be generalized to create clusters from other inorganic and polymer particles. We also show that high yields of particular morphologies can be obtained by precise control of the inorganic seed particle size, a finding that can be explained using a hard-sphere packing model. These clusters can be further chemically modified for a variety of applications. Introducing a cross-linker leads to colloidal clusters that can be index matched in an appropriate solvent, allowing them to be used for particle tracking or optical studies of colloidal self-assembly. Also, depositing a thin silica layer on these colloids allows the surface properties to be controlled using silane chemistry.

Design and synthesis of model transparent aqueous colloids with optimal scattering properties.

We demonstrate the synthesis and self-assembly of colloidal particles with independently controlled diameter and scattering cross section. We show that it is possible to prepare bulk colloidal suspensions that are nearly transparent in water, while the particles themselves can be individually resolved using optical microscopy. These particles may be ideal model colloids for real-space studies of self-assembly in aqueous media. Moreover, they illustrate the degree to which the optical properties of colloids can be engineered through straightforward chemistry.

New insights into the nucleation and growth of PS nodules on nanoparticles by 3D cryo-electron tomography.

The nucleation and growth of polystyrene (PS) nodules on 170 nm silica seeds under emulsion-polymerization conditions have been investigated for the first time by cryo-electron tomography. 3D arrangements were reconstructed from samples collected at several polymerization times (from 5 to 120 min). Early samples display the presence of small PS nodules bound to silica particles in a random distribution. For longer polymerization times, the number of PS nodules per silica seed decreases leading to octopod-like morphologies. The tomographic method allowed us to measure the contact angle that the growing PS nodules form with the silica bead surface. The average value of 142.4° remains constant over all of the observed period of the polymerization reaction. This contact angle appeared to be one of the key parameters for controlling the morphology of PS-silica biphasic particles.