Micellar Nanogels from Alginate-Based Diblock Copolysaccharides.

Alginates are marine polysaccharides known for their ability to selectively bind calcium ions and form hydrogels. They are widely used in biomedical applications but are challenging to produce as nanogels. Here we introduce a self-assembly route to create stable alginate-based nanogels under near-equilibrium conditions. Guluronate (G) blocks, which interact with divalent cations such as Ca2+, Ba2+, and Sr2+, were extracted from alginates and covalently linked through their reducing end to the reducing end of dextran (Dex) chains, forming linear block copolymers that self-assemble into micellar nanogels with a core-corona structure in the presence of these ions. Real-time dynamic light scattering (DLS) and small-angle neutron scattering (SANS) were used to study the self-assembly mechanism of the copolymer during dialysis against divalent ions. For the G12-b-Dex51 copolymer, we achieved spherical micelles with an 8 nm radius and an aggregation number of around 20. Although the type of divalent cation affected micelle stability, it did not influence their size. Micellar nanogels are dynamic structures, capable of ion exchange, and can disassemble with chelating agents like ethylenediamine tetraacetic acid (EDTA).

The desalting/salting pathway: a route to form metastable aggregates with tuneable morphologies and lifetimes.

We investigate the formation/re-dissociation mechanisms of hybrid complexes made from negatively charged PAA2k coated γ-Fe2O3 nanoparticles (NP) and positively charged polycations (PDADMAC) in aqueous solution in the regime of very high ionic strength (I). When the building blocks are mixed at large ionic strength (1 M NH4Cl), the electrostatic interaction is screened and complexation does not occur. If the ionic strength is then lowered down to a targeted ionic strength Itarget, there is a critical threshold Ic = 0.62 M at which complexation occurs, that is independent of the charge ratio Z and the pathway used to reduce salinity (drop-by-drop mixing or fast mixing). If salt is added back up to 1 M, the transition is not reversible and persistent out-of-equilibrium aggregates are formed. The lifetimes of such aggregates depends on Itarget: the closer Itarget to Ic is, the more difficult it is to dissolve the aggregates. Such peculiar behavior is driven by the inner structure of the complexes that are formed after desalting. When Itarget is far below Ic, strong electrostatic interactions induce the formation of dense, compact and frozen aggregates. Such aggregates can only poorly reorganize further on with time, which makes their dissolution upon resalting almost reversible. Conversely, when Itarget is close to Ic more open aggregates are formed due to weaker electrostatic interactions upon desalting. The system can thus rearrange with time to lower its free energy and reach more stable out-of-equilibrium states which are very difficult to dissociate back upon resalting, even at very high ionic strength.

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.

Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement.

The formulation pathway and/or the mixing method are known to be relevant in many out-of-equilibrium processes. In this work, we studied the effect of the mixing conditions on the physicochemical properties of poly-ε-caprolactone (PCL) particles prepared by solvent displacement. More specifically, water was added in one shot (fast addition) or drop by drop to PCL solution in tetrahydrofuran (THF) to study the impact of the mixing process on particle properties including size, stability, and crystallinity. Two distinct composition maps representing the Ouzo domain characteristic of the presence of metastable nanoparticles have been established for each mixing method. Polymer nanoparticles are formed in the Ouzo domain according to a nucleation and growth (or aggregation) mechanism. The fast addition promotes a larger nucleation rate, thus favoring the formation of small and uniform particles. For the drop-by-drop addition, for which the polymer solubility gradually decreases, the composition trajectories systematically cross an intermediate unstable region between the solubility limit of the polymer and the Ouzo domain. This leads to heterogeneous nucleation as shown by the formation of larger and less stable particles. Particles formed in the Ouzo domain have semi-crystalline properties. The PCL melting point is decreased with the THF fraction trapped in particles in accordance with Flory's theory for melt crystallization. On the other hand, the degree of crystallinity is constant, around 20% regardless of the THF fraction. No difference between fast and slow addition could be detected on the semi-crystalline properties of the particles which emphasize that thermodynamic rather than kinetic factors drive the polymer crystallization in particles. The recovery of bulk PCL crystallinity after the removal of THF from particles tends to confirm this hypothesis.

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.

Early stage kinetics of polyelectrolyte complex coacervation monitored through stopped-flow light scattering.

Polyelectrolyte complexes (PECs) between poly(acrylic acid) (PAA) and poly(diallyldimethylammonium chloride) (PDADMAC), a model system forming coacervate particles via electrostatic interaction at pH 10, were prepared by a stopped-flow (SF) fast mixing technique at different mixing charge ratios (z) and ionic strengths. Both PEC final morphologies prepared by either SF or manual one-shot mixing are similar at bench time. In situ light scattering combined with the SF technique pointed-out, however, the presence of three distinct early stage kinetic behaviors in the formation of PECs. The first stage observed at low z is ascribed to the relaxation/reorganization of soluble PECs. At higher z or in the presence of salt, a second stage is found corresponding to the aggregation and/or rearrangement of small soluble PECs into larger structures. Redistribution of excess charges among those PECs produces some neutral condensed coacervate droplets as well, coexisting with PECs in a wide range of mixing ratios. Finally, a last process featured with bell-shaped curves indicates the full coacervation that quickens while approaching charge neutrality and/or at higher ionic strength.

A Bacterial Myeloperoxidase with Antimicrobial Properties.

The four mammalian peroxidases (myeloperoxidase, eosinophilperoxidase, lactoperoxidase, and thyroid peroxidase) are widely studied in the literature. They catalyze the formation of antimicrobial compounds and participate in innate immunity. Owing to their properties, they are used in many biomedical, biotechnological, and agro-food applications. We decided to look for an enzyme that is easiest to produce and much more stable at 37 °C than mammalian peroxidases. To address this question, a peroxidase from Rhodopirellula baltica, identified by bioinformatics tools, was fully characterized in this study. In particular, a production and purification protocol including the study of heme reconstitution was developed. Several activity tests were also performed to validate the hypothesis that this peroxidase is a new homolog of mammalian myeloperoxidase. It has the same substrate specificities as the human one and accepts I-, SCN-, Br-, and Cl- as (pseudo-) halides. It also exhibits other auxiliary activities such as catalase and classical peroxidase activities, and it is very stable at 37 °C. Finally, this bacterial myeloperoxidase can kill the Escherichia coli strain ATCC25922, which is usually used to perform antibiograms.

In Situ Monitoring of Block Copolymer Self-Assembly via Solvent Exchange through Controlled Dialysis with Light and Neutron Scattering Detection.

Solution self-assembly of amphiphilic block copolymers (BCs) is typically performed by a solvent-to-water exchange. However, BC assemblies are often trapped in metastable states depending on the mixing conditions such as the magnitude and rate of water addition. BC self-assembly can be performed under near thermodynamic control by dialysis, which accounts for a slow and gradual water addition. In this Letter we report the use of a specifically designed dialysis cell to continuously monitor by dynamic light scattering and small-angle neutron scattering the morphological changes of PDMS-b-PEG BCs self-assemblies during THF-to-water exchange. The complete phase diagrams of near-equilibrium structures can then be established. Spherical micelles first form before evolving to rod-like micelles and vesicles, decreasing the total developed interfacial area of self-assembled structures in response to increasing interfacial energy as the water content increases. The dialysis kinetics can be tailored to the time scale of BC self-assembly by modifying the membrane pore size, which is of interest to study the interplay between thermodynamics and kinetics in self-assembly pathways.

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-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.

Complexation in Aqueous Solution of a Hydrophobic Polyanion (PSSNa) Bearing Different Charge Densities with a Hydrophilic Polycation (PDADMAC).

In this work the electrostatic complexation of two strong polyelectrolytes (PEs) was studied, the hydrophilic and positively charged poly (diallyldimethylammonium chloride) (PDADMAC) and the hydrophobic and negatively charged poly (styrene-co-sodium styrene sulfonate) (P(St-co-SSNa)), which was prepared at different sulfonation rates. The latter is known to adopt a pearl necklace conformation in solution for intermediate sulfonation rates, suggesting that a fraction of the P(St-co-SSNa) charges might be trapped in these hydrophobic domains; thus making them unavailable for complexation. The set of complementary techniques (DLS, zetametry, ITC, binding experiment with a cationic and metachromatic dye) used in this work highlighted that this was not the case and that all anionic charges of P(St-co-SSNa) were in fact available for complexation either with the polycationic PDADMAC or the monocationic o-toluidine blue dye. Only minor differences were observed between these techniques, consistently showing a complexation stoichiometry close to 1:1 at the charge equivalence for the different P(St-co-SSNa) compositions. A key result emphasizing that (i) the strength of the electrostatic interaction overcomes the hydrophobic effect responsible for pearl formation, and (ii) the efficiency of complexation does not depend significantly on differences in charge density between PDADMAC and P(St-co-SSNa), highlighting that PE chains can undergo conformational rearrangements favoring the juxtaposition of segments of opposite charge. Finally, these data have shown that the formation of colloidal PECs, such as PDADMAC and P(St-co-SSNa), occurs in two distinct steps with the formation of small primary complex particles (<50 nm) by pairing of opposite charges (exothermic step) followed by their aggregation within finite-size clusters (endothermic step). This observation is in agreement with the previously described mechanism of PEC particle formation from strongly interacting systems containing a hydrophobic PE.

Hybrid polymeric micelles stabilized by gallium ions: Structural investigation.

The addition of gallium ions to a solution of a double-hydrophilic block copolymer, i.e. poly(ethylene oxide)-block-poly(acrylic acid), leads to the spontaneous formation of highly monodisperse micelles with a Hybrid PolyIon Complexes (HPICs) core. By combining several techniques, a precise description of the HPIC architecture was achieved. In particular and for the first time, NMR and anomalous small angle X-ray scattering (ASAXS) enable tracking of the inorganic ions in solution and highlighting the co-localization of the gallium and the poly(acrylic acid) blocks in a rigid structure at the core of the micelle. Such a core has a radius of ca 4.3 nm while the complete nano-object with its poly(ethylene oxide) shell has a total radius of ca 11 nm. The aggregation number was also estimated using the ASAXS results. This comprehensive structural characterization of the Ga HPICs corroborates the assumptions made for HPICs based on other inorganic ions and demonstrates the universality of the HPIC structure leading, for example, to new families of contrast agents in medical imaging.

Interfacial Behavior of Solid- and Liquid-like Polyelectrolyte Complexes as a Function of Charge Stoichiometry.

We systematically investigate in this work the surface activity of polyelectrolyte complex (PECs) suspensions as a function of the molar charge ratio Z (= [-]/[+]) from two model systems: the weakly and strongly interacting poly (diallyldimethylammonium chloride)/poly (acrylic acid sodium salt) (PDADMAC/PANa) and poly (diallyldimethylammonium chloride)/poly (sodium 4- styrenesulfonate) (PDADMAC/PSSNa) pairs, respectively. For both systems, the PEC surface tension decreases as the system approaches charge stoichiometry (Z = 1) whenever the complexation occurs in the presence of excess PDADMAC (Z < 1) or excess polyanion (Z > 1) consistent with an increased level of charge neutralization of PEs forming increasingly hydrophobic and neutral surface-active species. The behavior at stoichiometry (Z = 1) is also particularly informative about the physical nature of the complexes. The PDADMAC/PANa system undergoes a liquid-liquid phase transition through the formation of coacervate microdroplets in equilibrium with macroions remaining in solution. In the PDADMAC/PSSNa system, the surface tension of the supernatant was close to that of pure water, suggesting that the PSSNa-based complexes have completely sedimented, consistent with a complete liquid-solid phase separation of an out-of-equilibrium system. Besides, the high sensitivity of surface tension measurements, which can detect the presence of trace amounts of aggregates and other precursors in the supernatant, allows for very accurate determination of the exact charge stoichiometry of the complexes. Finally, the very low water/water interfacial tension that develops between the dilute phase and the denser coacervate phase in the PDADAMAC/PANa system was measured using the generalized Young-Laplace method to complete the full characterization of both systems. The overall study showed that simple surface tension measurements can be a very sensitive tool to characterize, discriminate, and better understand the formation mechanism of the different structures encountered during the formation of PECs.

Exploration of space to achieve scientific breakthroughs.

Living organisms adapt to changing environments using their amazing flexibility to remodel themselves by a process called evolution. Environmental stress causes selective pressure and is associated with genetic and phenotypic shifts for better modifications, maintenance, and functioning of organismal systems. The natural evolution process can be used in complement to rational strain engineering for the development of desired traits or phenotypes as well as for the production of novel biomaterials through the imposition of one or more selective pressures. Space provides a unique environment of stressors (e.g., weightlessness and high radiation) that organisms have never experienced on Earth. Cells in the outer space reorganize and develop or activate a range of molecular responses that lead to changes in cellular properties. Exposure of cells to the outer space will lead to the development of novel variants more efficiently than on Earth. For instance, natural crop varieties can be generated with higher nutrition value, yield, and improved features, such as resistance against high and low temperatures, salt stress, and microbial and pest attacks. The review summarizes the literature on the parameters of outer space that affect the growth and behavior of cells and organisms as well as complex colloidal systems. We illustrate an understanding of gravity-related basic biological mechanisms and enlighten the possibility to explore the outer space environment for application-oriented aspects. This will stimulate biological research in the pursuit of innovative approaches for the future of agriculture and health on Earth.

Microfluidics with on-line dynamic light scattering for size measurements.

We present a detailed investigation on the feasibility of on-line dynamic light scattering measurements of colloidal sizes in a pressure-driven microfluidic flow. We review some theoretical arguments showing that such experiments are difficult to perform due to the Poiseuille flow that induces interferences of different Doppler shifts. Such a theoretical approach is however very useful to figure out the range of parameters where on-line size measurements are possible. We then build a dynamic light scattering setup around a microfluidic chip that enables us to estimate the size of Brownian scatterers flowing in PDMS-based microchannels, thus validating experimentally the theoretical estimations. We finally present a microfluidic chip that can mix two reactants in approximately 200 ms, and allows size measurements using dynamic light scattering at about 300 ms after complete mixing. Two applications are presented: the continuous monitoring of the viscosity of a two-fluid mixture, and the electrostatic co-assembly of oppositely charged nanoparticles and block copolymers.

Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with siRNA.

In the context of gene delivery, chitosan has been widely used as a safe and effective polycation to complex DNA, RNA and more recently, siRNA. However, much less attention has been paid to chitosan oligosaccharides (COS) despite their biological properties. This study proposed to carry out a physicochemical study of COS varying in degree of polymerization (DP) from 5 to 50, both from the point of view of the solution properties and the complexing behavior with siRNA. The main parameters studied as a function of DP were the apparent pKa, the solubility versus pH, the binding affinity with siRNA and the colloidal properties of complexes. Some parameters, like the pKa or the binding enthalpy with siRNA, showed a marked transition from DP 5 to DP 13, suggesting that electrostatic properties of COS vary considerably in this range of DP. The colloidal properties of siRNA/COS complexes were affected in a different way by the COS chain length. In particular, COS of relatively high DP (≥50) were required to form small complex particles with good stability.

Organic versus hybrid coacervate complexes: co-assembly and adsorption properties.

We report the co-assembly and adsorption properties of coacervate complexes made from polyelectrolyte-neutral block copolymers and oppositely charged nanocolloids. The nanocolloids put under scrutiny were ionic surfactant micelles and highly charged 7 nm cerium oxide (CeO2) nanoparticles. Static and dynamic light scattering was used to investigate the microstructure and stability of the organic and hybrid complexes. For five different systems of nanocolloids and polymers, we first demonstrated that the electrostatic complexation resulted in the formation of stable core-shell aggregates in the 100 nm range. The microstructure of the CeO2-based complexes was resolved using cryogenic transmission electronic microscopy (Cryo-TEM), and it revealed that the cores were clusters made from densely packed nanoparticles, presumably through complexation of the polyelectrolyte blocks by the surface charges. The cluster stability was monitored by systematic light scattering measurements. In the concentration range of interest, c = 10-4-1 wt.%, the surfactant-based complexes were shown to exhibit a critical association concentration (cac) whereas the nanoparticle-polymer hybrids did not. The adsorption properties of the same complexes were investigated above the cac by stagnation point adsorption reflectometry. The adsorbed amount was measured as a function of time for polymers and complexes using anionically charged silica and hydrophobic poly(styrene) substrates. It was found that all complexes adsorbed readily on both types of substrates up to a level of 1-2 mg m-2 at stationary state. Upon rinsing however, the adsorbed layer was removed for the surfactant-based systems, but not for the cerium oxide clusters. As for the solution properties, these finding were interpreted in terms of a critical association concentrations, which are very different for organic and hybrid complexes. Combining the efficient adsorption and strong stability of the CeO2-based core-shell hybrids on various substrates, it is finally suggested that these systems could be used appropriately for coating and anti-biofouling applications.

Live nanoscopic to mesoscopic topography reconstruction with an optical microscope for chemical and biological samples.

Macroscopic properties of physical and biological processes like friction, wetting, and adhesion or cell migration are controlled by interfacial properties at the nanoscopic scale. In an attempt to bridge simultaneously investigations at different scales, we demonstrate here how optical microscopy in Wet-Surface Ellipsometric Enhanced Contrast (Wet-SEEC) mode offers imaging and measurement of thin films at solid/liquid interfaces in the range 1-500 nm with lateral optical resolution. A live, label-free and noninvasive methodology integrated with microfluidic devices allowed here characterization of polymers and proteins patterns together with corresponding phenotypes of living cells.

Structure, thermodynamic and kinetic signatures of a synthetic polyelectrolyte coacervating system.

While many studies on coacervation have targeted biomacromolecules, we review in this article the key structure, thermodynamic and kinetic features of a fully synthetic coacervating system based on polyacrylic acid (PAA) and poly(diallyldimethylammonium chloride) (PDADMAC) oppositely charged polyelectrolytes at pH10, where PAA chains are fully deprotonated. Among the main points of interest, we can highlight (i) the presence of polyelectrolyte complex (PEC) nanoparticles that, unexpectedly, coexist with a certain amount of coacervate droplets in a large range of compositions, even far from stoichiometry; (ii) the fact that these PEC nanoparticles are likely precursors of the coacervation occurring at stoichiometry; (iii) the formation of soluble PECs only in a certain range of physicochemical conditions; (iv) the equilibrium properties of the system; (v) and last but not least a distinctive kinetic signature at stoichiometry evidenced by a peak in light scattering at very short times (~100ms). Some of these results can be rationalized on the basis of weak interaction unfolding between oppositely charged PAA and PDADMAC chains as revealed by microcalorimetry measurements.

Ions-induced nanostructuration: effect of specific ionic adsorption on hydrophobic polymer surfaces.

The effect of surface charges on the ionic distribution in close proximity to an interface has been extensively studied. On the contrary, the influence of ions (from dissolved salts) on deformable interfaces has been barely investigated. Ions can adsorb from aqueous solutions on hydrophobic surfaces, generating forces that can induce long-lasting deformation of glassy polymer films, a process called ion-induced polymer nanostructuration, IPN. We have found that this process is ion-specific; larger surface modifications are observed in the presence of water ions and hydrophobic and amphiphilic ions. Surface structuration is also observed in the presence of certain salts of lithium. We have used streaming potential and atomic force microscopy to study the effect of dissolved ions on the surface properties of polystyrene films, finding a good correlation between ionic adsorption and IPN. Our results also suggest that the presence of strongly hydrated lithium promotes the interaction of anions with polystyrene surfaces and more generally with hydrophobic polymer surfaces, triggering then the IPN process.