12-hydroxystearic acid-mediated in-situ surfactant generation: A novel approach for organohydrogel emulsions.

HYPOTHESIS: Organohydrogel emulsions display unique rheological properties and contain hydrophilic and lipophilic domains highly desirable for the loading of active compounds. They find utility in various applications from food to pharmaceuticals and cosmetic products. The current systems have limited applications due to complex expensive formulation and/or processing difficulties in scale-up. To solve these issues, a simple emulsification process coupled with unique compounds are required. EXPERIMENTS: Here, we report an organohydrogel emulsion based only on a low concentration of 12-hydroxystearic acid acting as a gelling agent for both oil and water phases but also as a surfactant. The emulsification process is based on in-situ surfactant transfer. We characterize the emulsification process occurring at the nanoscale by using tensiometry experiments. The emulsion structure was determined by coupling Small Angle X-ray and neutron scattering, and confocal Raman microscopy. FINDINGS: We demonstrate that the stability and unique rheological properties of these emulsions come from the presence of self-assembled crystalline structures of 12-hydroxystearic acid in both liquid phases. The emulsion properties can be tuned by varying the emulsion composition over a wide range. These gelled emulsions are prepared using a low energy method offering easy scale-up at an industrial level.

One step generation of single-core double emulsions from polymer-osmose-induced aqueous phase separation in polar oil droplets.

Water-in-oil-in-water emulsions (W/O/W) are aqueous droplet(s) embedded within oil droplets dispersed in a continuous water phase. They are attracting interest due to their possible applications from cosmetic to food science since both hydrosoluble and liposoluble cargos can be encapsulated within. They are generally prepared using a one-step or a two-step method, phase inversion and also via spontaneous emulsification. Here, we describe a general and simple one-step method based on hydrophilic polymers dispersed in polar oils to generate osmose-induced diffusion of water into oil droplets, forming polymer-rich aqueous droplets inside the oil droplets. Polyethylene glycol, but also other hydrophilic polymers (branched polyethylene imine or polyvinyl pyrrolidone) were successfully dispersed in 1-octanol or other polar oils (oleic acid or tributyrin) to produce an O/W emulsion that spontaneously transformed into a W1/O/W2 emulsion, with the inner aqueous droplet (W1) only containing the hydrophilic polymer initially dispersed in oil. By combining single drop experiments, with macroscopic viscosity measurements, we demonstrated that the double emulsion resulted of water diffusion, which amplitude could be adjusted by the polymer concentration. The production of high internal phase emulsions was also achieved, together with a pH-induced transition from multiple to single core double emulsion. We expect this new method for producing double emulsions to find applications in domains of microencapsulation and materials chemistry.

Double Emulsion Droplets as a Plausible Step to Fatty Acid Protocells.

It is assumed that life originated on the Earth from vesicles made of fatty acids. These amphiphiles are the simplest chemicals, which can be present in the prebiotic soup, capable of self-assembling into compartments mimicking modern cells. Production of fatty acid vesicles is widely studied, as their growing and division. However, how prebiotic chemicals require to further yield living cells encapsulated within protocells remains unclear. Here, one suggests a scenario based on recent studies, which shows that phospholipid vesicles can form from double emulsions affording facile encapsulation of cargos. In these works, water-in-oil-in-water droplets are produced by microfluidics, having dispersed lipids in the oil. Dewetting of the oil droplet leaves the internal aqueous droplet covered by a lipid bilayer, entrapping cargos. In this review, formation of fatty acid protocells is briefly reviewed, together with the procedure for preparing double emulsions and vesicles from double emulsion and finally, it is proposed that double emulsion droplets formed in the deep ocean where undersea volcano expulsed materials, with fatty acids (under their carboxylic form) and alkanols as the oily phase, entrapping hydrosoluble prebiotic chemicals in a double emulsion droplet core. Once formed, double emulsion droplets can move up to the surface, where an increase of pH, variation of pressure and/or temperature may have allowed dewetting of the oily droplet, leaving a fatty acid vesicular protocell with encapsulated prebiotic materials.

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.

Cytoskeletal components can turn wall-less spherical bacteria into kinking helices.

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Stabilization of all-aqueous droplets by interfacial self-assembly of fatty acids bilayers.

All-aqueous microdroplets produced by liquid-liquid phase separation have emerged as promising models of artificial cells, and offer new approaches for the solvent-free encapsulation of fragile solutes. Yet, the lack of a membrane on such droplets makes them intrinsically unstable against coarsening, and precludes a fine control over chemical localization, as solutes can freely diffuse through the interface. Herein, we report the construction of stable and impermeable water-in-water emulsions via the interfacial self-assembly of mixed sodium oleate/1-decanol bilayers on dextran-rich droplets produced by segregative liquid-liquid phase separation with poly(ethylene glycol). Lipids spontaneously self-assemble as multilamellar structures at the surface of the droplets as revealed by freeze-fracture transmission electron microscopy and small-angle X-ray scattering. We further demonstrate that the lipid-based membrane is impermeable to oligonucleotides and proteins, but also to a low molecular weight dye, so that a strict chemical encapsulation can be achieved by spontaneous partitioning within the droplets before membrane self-assembly. Taken together, our results highlight the ease of production of fatty acid-stabilized all-aqueous emulsions droplets able to encapsulate a range of solutes without the need of oil or organic solvents, paving the way to the construction of robust membrane-bounded, polymer-rich artificial cells.

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.

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.

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.

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.

Antagonistic chemical coupling in self-reconfigurable host-guest protocells.

Fabrication of compartmentalised chemical systems with nested architectures and biomimetic properties has important implications for controlling the positional assembly of functional components, spatiotemporal regulation of enzyme cascades and modelling of proto-organelle behaviour in synthetic protocells. Here, we describe the spontaneous capture of glucose oxidase-containing proteinosomes in pH-sensitive fatty acid micelle coacervate droplets as a facile route to multi-compartmentalised host-guest protocells capable of antagonistic chemical and structural coupling. The nested system functions co-operatively at low-substrate turnover, while high levels of glucose give rise to pH-induced disassembly of the droplets, release of the incarcerated proteinosomes and self-reconfiguration into spatially organised enzymatically active vesicle-in-proteinosome protocells. Co-encapsulation of antagonistic enzymes within the proteinosomes produces a sequence of self-induced capture and host-guest reconfiguration. Taken together, our results highlight opportunities for the fabrication of self-reconfigurable host-guest protocells and provide a step towards the development of protocell populations exhibiting both synergistic and antagonistic modes of interaction.

Preparation of Swellable Hydrogel-Containing Colloidosomes from Aqueous Two-Phase Pickering Emulsion Droplets.

The fabrication of stable colloidosomes derived from water-in-water Pickering-like emulsions are described that were produced by addition of fluorescent amine-modified polystyrene latex beads to an aqueous two-phase system consisting of dextran-enriched droplets dispersed in a PEG-enriched continuous phase. Addition of polyacrylic acid followed by carbodiimide-induced crosslinking with dextran produces hydrogelled droplets capable of reversible swelling and selective molecular uptake and exclusion. Colloidosomes produced specifically in all-water systems could offer new opportunities in microencapsulation and the bottom-up construction of synthetic protocells.

Catanionic Coacervate Droplets as a Surfactant-Based Membrane-Free Protocell Model.

We report on the formation of surfactant-based complex catanionic coacervate droplets in mixtures of decanoic acid and cetylpyridinium chloride or cetyltrimethylammonium bromide. We show that coacervation occurs over a broad range of composition, pH, and ionic strength. The catanionic coacervates consist of elongated micelles, sequester a wide range of solutes including water-soluble organic dyes, polysaccharides, proteins, enzymes, and DNA, and can be structurally stabilized by sodium alginate or gelatin-based hydrogelation. These results suggest that catanionic coacervates could be exploited as a novel surfactant-based membrane-free protocell model.

Sequestration of Proteins by Fatty Acid Coacervates for Their Encapsulation within Vesicles.

Encapsulating biological materials in lipid vesicles is of interest for mimicking cells; however, except in some particular cases, such processes do not occur spontaneously. Herein, we developed a simple and robust method for encapsulating proteins in fatty acid vesicles in high yields. Fatty acid based, membrane-free coacervates spontaneously sequester proteins and can reversibly form membranous vesicles upon varying the pH value, the precrowding feature in coacervates allowing for protein encapsulation within vesicles. We then produced enzyme-enriched vesicles and show that enzymatic reactions can occur in these micrometric capsules. This work could be of interest in the field of synthetic biology for building microreactors.

Clouding in fatty acid dispersions for charge-dependent dye extraction.

The clouding phenomenon in non-ionic surfactant systems is a common feature that remains rare for ionic detergents. Here, we show that fatty acid (negatively charged) systems cloud upon cooling hot dispersions depending on the concentration or when adding excess guanidine hydrochloride. The clouding of these solutions yields the formation of enriched fatty acid droplets in which they exhibit a polymorphism that depends on the temperature: upon cooling, elongated wormlike micelles transit to rigid stacked bilayers inside droplets. Above this transition temperature, droplets coalesce yielding a phase separation between a fatty acid-rich phase and water, allowing extraction of dyes depending on their charge and lipophilicity. Positively charged and zwitterionic dyes were sequestered within the droplets (and then in the fatty acid-rich upper phase) whereas the negatively charged ones were found in both phases. Our results show an additional case of negatively charged surfactant which exhibit clouding phenomenon and suggest that these systems could be used for extracting solutes depending on their charge and lipophilicity.

Self-Assembly of Bilayer Vesicles Made of Saturated Long Chain Fatty Acids.

Saturated long chain fatty acids (sLCFA, e.g., C14:0, C16:0, and C18:0) are potentially the greenest and cheapest surfactants naturally available. However, because aqueous sodium soaps of sLCFA are known to crystallize, the self-assembly of stable bilayer vesicles has not been reported yet. Here, by using such soaps in combination with guanidine hydrochloride (GuHCl), which has been shown recently to prevent crystallization, we were capable of producing stable bilayer vesicles made of sLCFA. The phase diagrams were established for a variety of systems showing that vesicles can form in a broad range of composition and pH. Both solid state NMR and small-angle neutron scattering allowed demonstrating that in such vesicles sLCFA are arranged in a bilayer structure which exhibits similar dynamic and structural properties as those of phospholipid membranes. We expect these vesicles to be of interest as model systems of protocells and minimal cells but also for various applications since fatty acids are potentially substitutes to phospholipids, synthetic surfactants, and polymers.

Self-assembly of myristic acid in the presence of choline hydroxide: effect of molar ratio and temperature.

Salt-free catanionic systems based on fatty acids exhibit a broad polymorphism by simply tuning the molar ratio between the two components. For fatty acid combined with organic amino counter-ions, very few data are available on the phase behavior obtained as a function of the molar ratio between the counter-ion and the fatty acid. We investigated the choline hydroxide/myristic acid system by varying the molar ratio, R=n(choline hydroxide)/n(myristic acid), and the temperature. Myristic acid ionization state was determined by coupling pH, conductivity and infra-red spectroscopy measurements. Self-assemblies were characterized by small angle neutron scattering and microscopy experiments. Self-assembly thermal behavior was investigated by differential scanning calorimetry, wide angle X-ray scattering and nuclear magnetic resonance. For R<1, ionized and protonated myristic acid molecules coexisted leading to the formation of facetted self-assemblies and lamellar phases. The melting process between the gel and the fluid state of these bilayers induced a structural change from facetted or lamellar objects to spherical vesicles. For R>1, myristic acid molecules were ionized and formed spherical micelles. Our study highlights that both R and temperature are two key parameters to finely control the self-assembly structure formed by myristic acid in the presence of choline hydroxide.

Aminosilane/oleic acid vesicles as model membranes of protocells.

Oleic acid vesicles represent good models of membrane protocells that could have existed in prebiotic times. Here, we report the formation, growth polymorphism, and dynamics of oleic acid spherical vesicles (1-10 µm), stable elongated vesicles (>50 µm length; 1-3 µm diameter), and chains of vesicles (pearl necklaces, >50 µm length; 1-3 µm diameter) in the presence of aminopropyl triethoxysilane and guanidine hydrochloride. These vesicles exhibit a remarkable behavior with temperature: spherical vesicles only are observed when keeping the sample at 4 °C for 2 h, and self-aggregated spherical vesicles occur upon freezing/unfreezing (-20/20 °C) samples. Rather homogeneous elongated vesicles are reformed upon heating samples at 80 °C. The phenomenon is reversible through cycles of freezing/heating or cooling/heating of the same sample. Deuterium NMR evidences a chain packing rigidity similar to that of phospholipid bilayers in cellular biomembranes. We expect these bilayered vesicles to be surrounded by a layer of aminosilane oligomers, offering a variant model for membrane protocells.

Fully deuterated magnetically oriented system based on fatty acid direct hexagonal phases.

There is strong demand in the field of NMR for simple oriented lipid supramolecular assemblies, the constituents of which can be fully deuterated, for specifically studying the structure of host protonated molecules (e.g., peptides, proteins...) in a lipid environment. Also, small-angle neutron scattering (SANS) in fully deuterated oriented systems is powerful for gaining information on protonated host molecules in a lipid environment by using the contrast proton/deuterium method. Here we report on a very simple system made of fatty acids (dodecanoic and tetradecanoic) and ethanolamine in water. All components of this system can be obtained commercially as perdeuterated. Depending on the molar ratio and the concentration, the system self-assembles at room temperature into a direct hexagonal phase that is oriented by moderate magnetic fields of a few tesla. The orientation occurs within the magnetic field upon cooling the system from its higher-temperature isotropic phase: the lipid cylinders of the hexagonal phase become oriented parallel to the field. This is shown by solid-state NMR using either perdeuterated fatty acids or ethanolamine. This system bears strong interest for studying host protonated molecules but also in materials chemistry for building oriented solid materials.

Polymorphism of natural fatty acid liquid crystalline phases.

We study the phase behavior in water of a mixture of natural long chain fatty acids (FAM) in association with ethylenediamine (EDA) and report a rich polymorphism depending on the composition. At a fixed EDA/FAM molar ratio, we observe upon dilution a succession of organized phases going from a lamellar phase to a hexagonal phase and, finally, to cylindrical micelles. The phase structure is established using polarizing microscopy, SAXS, and SANS. Interestingly, in the lamellar phase domain, we observe the presence of defects upon dilution, which SAXS shows to correspond to intrabilayer correlations. NMR and FF-TEM techniques suggest that these defects are related to an increase in the spontaneous curvature of the molecule monolayers in the lamellae. ATR-FTIR spectroscopy was also used to investigate the degree of ionization within these assemblies. The successive morphological transitions are discussed with regards to possible molecular mechanisms, in which the interaction between the acid surfactant and the amine counterion plays the leading role.