Freezing-mediated formation of supraproteins using depletion forces.

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein-polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein-polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Effects of polymer nonideality on depletion-induced phase behaviour of colloidal disks and rods.

Colloidal dispersions composed of either platelets or rods exhibit liquid crystalline phase behaviour that is strongly influenced by the addition of nonadsorbing polymers. In this work we examined how polymer segment-segment interactions affect this phase behaviour as compared to using either penetrable hard spheres (PHS) or ideal ('ghost') chains as depletants. We find that the simplified polymer description predicts the same phase diagram topologies as the more involved polymer descriptions. Therefore the PHS description is still adequate for qualitative predictions. For sufficiently large polymer sizes we find however that the precise polymer description significantly alters the locations of the phase coexistence regions. Especially the stability region of isotropic-isotropic coexistence is affected by the polymer interactions. To illustrate the quantitative effects some examples are presented.

Phase stability of colloidal spheres mixed with semiflexible supramolecular polymers.

HYPOTHESIS: Mixtures of colloids and supramolecular polymers may exhibit stimuli-responsive phase behaviour. However, in theoretical descriptions of such systems, the polymers are commonly described either as flexible chains or as rigid rods, while in experimental systems supramolecular polymers usually fall in between these two limits. We expect the flexibility of the polymers to have a profound effect on the stimuli-responsive phase behaviour. THEORY: We propose a general approach to predict the phase behaviour of colloidal hard spheres mixed with covalent or supramolecular polymers of arbitrary persistence length using free volume theory and an interpolation between flexible and rigid chains. FINDINGS: The binodals are predicted to shift to lower monomer concentrations as the persistence length is increased, making the polymers more efficient depletants. The persistence length is therefore an additional degree of freedom for manipulating the phase behaviour of colloid-polymer mixtures. We show that by manipulating the persistence length of temperature responsive supramolecular polymers, a wide range of phase diagrams with various topologies can be obtained. For example, we find phase diagrams with a critical point but no triple point or displaying two triple points for temperature-sensitive supramolecular polymers mixed with hard spheres.

Multiphase Coexistences in Rod-Polymer Mixtures.

Using recently derived analytical equations of state for hard rod dispersions, we predict the phase behavior of athermal rod-polymer mixtures with free volume theory. The rods are modeled as hard spherocylinders, while the nonadsorbing polymer chains are described as penetrable hard spheres. It is demonstrated that all of the different types of phase states that are stable for pure colloidal rod dispersions can coexist with any combination of these phases if polymers are added, depending on the concentrations, rod aspect ratio, and polymer-rod size ratio. This includes novel two-, three-, and four-phase coexistences and isostructural coexistences between dilute and concentrated phases of the same kind, even for the more ordered (liquid) crystal phases. This work provides insight into the conditions at which particular multiphase coexistences are expected for well-defined model colloidal rod-polymer mixtures. We provide a quantitative map detailing the various types of isostructural coexistences, which confirms an early qualitative hypothesis by Bolhuis et al. ( J. Chem. Phys. 107, 1997 1551).

From a eutectic mixture to a deep eutectic system via anion selection: Glutaric acid + tetraethylammonium halides.

In pursuit of understanding structure-property relationships for the melting point depression of binary eutectic mixtures, the influence of the anion on the solid-liquid (S-L) phase behavior was explored for mixtures of glutaric acid + tetraethylammonium chloride, bromide, and iodide. A detailed experimental evaluation of the S-L phase behavior revealed that the eutectic point is shifted toward lower temperatures and higher salt contents upon decreasing the ionic radius. The salt fusion properties were experimentally inaccessible owing to thermal decomposition. The data were inter- and extrapolated using various models for the Gibbs energy of mixing fitted to the glutaric-acid rich side only, which allowed for the assessment of the eutectic point. Fitting the experimental data to a two-parameter Redlich-Kister expansion with Flory entropy, the eutectic depth could be related to the ionic radius of the anion. The anion type, and in particular its size, can therefore be viewed as an important design parameter for the liquid window of other acid and salt-based deep eutectic solvents/systems.

Repulsive and attractive depletion forces mediated by nonadsorbing polyelectrolytes in the Donnan limit.

In mixtures of colloids and nonadsorbing polyelectrolytes, a Donnan potential arises across the region between surfaces that are depleted of the polyelectrolyte and the rest of the system. This Donnan potential tends to shift the polyelectrolyte density profile toward the colloidal surface and leads to the local accumulation of polyelectrolytes. We derive a zero-field theory for the disjoining pressure between two parallel flat plates. The polyelectrolyte is allowed to enter the confined interplate region at the cost of a conformational free energy penalty. The resulting disjoining pressure shows a crossover to a repulsive regime when the interplate separation gets smaller than the size of the polyelectrolyte chain, followed by an attractive part. We find a quantitative match between the model and self-consistent field computations that take into account the full Poisson-Boltzmann electrostatics.

Solvent Selectivity Governs the Emergence of Temperature Responsiveness in Block Copolymer Self-Assembly.

In highly selective solvents, block copolymers (BCPs) form association colloids, while in solvents with poor selectivity, they exhibit a temperature-controlled (de)mixing behavior. Herein, it is shown that a temperature-responsive self-assembly behavior emerges in solvent mixtures of intermediate selectivity. A biocompatible poly-ethylene(oxide)-block-poly-ε-caprolactone (PEO-PCL) BCP is used as a model system. The polymer is dissolved in solvent mixtures containing water (a strongly selective solvent for PEO) and ethanol (a poorly selective solvent for PEO) to tune the solvency conditions. Using synchrotron X-ray scattering, cryogenic transmission electron microscopy, and scanning probe microscopy, it is shown that a rich temperature-responsive behavior can be achieved in certain solvent mixtures. Crystallization of the PCL block enriches the phase behavior of the BCP by promoting sphere-to-cylinder morphology transitions at low temperatures. Increasing the water fraction in the solvent causes a suppression of the sphere-to-cylinder morphology transition. These results open up the possibility to induce temperature-responsive properties on demand in a wide range of BCP systems.

Quantification of the Structure of Colloidal Gas-Liquid Interfaces.

We have quantified the structure of the colloidal gas-liquid interface using synchrotron X-ray reflectivity measurements on a model colloid-polymer mixture. The interfacial width shows mean-field scaling with the colloid density difference, and the density profiles appear to be monotonic. Furthermore, our measurements allow us to distinguish between different theoretical polymer descriptions commonly used to model colloid-polymer mixtures. Our results highlight the importance of capturing the correct polymer physics in obtaining a quantitative theoretical description of the colloidal gas-liquid interface.

Design of Nonideal Eutectic Mixtures Based on Correlations with Molecular Properties.

In this work, a statistical analysis was performed to reveal how the molecular properties are correlated with the nonideal behavior observed in eutectic mixtures. From this, a statistical model, combined with theory and experimental results, was developed to predict the nonideal behavior of a specific set of eutectic mixtures, consisting of quaternary ammonium bromides with dicarboxylic acids and polyols. The combination of this analysis and this model can be considered as a first step toward the a priori design of eutectic mixtures. The analysis performed is based on principal components. The descriptors used for this are molecular properties of the constituents of these mixtures. The molecular properties are a combination of experimental, theoretical, and computed properties. The analysis reveals that there are strong correlations between the nonideality of the mixtures and a measure of the acidity of the hydrogen bond donating protons, the displacement of the bromide anion, and the bulkiness of the quaternary ammonium salt. Our analysis highlights the design rules of deep eutectic systems (DES), enabling control over the extent of the liquid window. Our model enables prediction of the eutectic temperature for a range of related mixtures.

Oil-in-water emulsions based on hydrophobic eutectic systems.

We demonstrate that oil-in-water emulsions can be prepared from hydrophobic eutectic systems (ES). Light microscopy and dynamic light scattering show that droplets are formed and zeta potential measurements indicate sufficient stability against coalescence. We investigate whether Ostwald ripening occurs in these ES-in-water emulsions by following the droplet growth over time and comparing it with an emulsion comprising decane in water. At first sight, the Ostwald ripening rate of the ES-in-water emulsion is expected to be orders of magnitude larger than the ripening of the decane-in-water emulsion due to a much higher solubility of the dispersed phase. However, experimentally we find that the ES-in-water emulsion actually grows a factor of two slower than the decane-in-water emulsion. We attribute this to the two-component nature of the ES, since the growth rate is mainly set by the least-soluble component of the ES. Thus, ESs offer the advantage of creating liquid emulsions of solid components, while setting the emulsion stability through their composition.

Polymer-mediated colloidal stability: on the transition between adsorption and depletion.

Addition of polymers to a colloidal dispersion modulates the interactions between the colloids. We briefly review the effects of positive and negative adsorption (also termed depletion). The effective colloid-polymer interactions sensitively affect the colloidal phase behavior. We present a theoretical framework to predict the phase behavior of colloid-polymer mixtures for varying affinities between colloid and polymer, leading to either positive or negative adsorption of polymer segments. For certain conditions, polymers are neither depleted nor adsorbed: the polymer concentration is essentially constant up to the colloidal surface, a condition which we term neutral adsorption. Near this condition, the calculated phase diagrams reveal a stable-unstable-restabilisation transition with increasing polymer concentration. Similar effects have been reported experimentally, for instance as a function of temperature [Feng et al., Nat. Mat., 2015, 14, 61-65], which may modulate the effective polymer-colloid affinity. Understanding how to achieve neutral adsorption opens up the possibility of preparing highly dense, yet stable, colloid-polymer mixtures.

Experimental Evidence for Algebraic Double-Layer Forces.

According to conventional wisdom, electric double-layer forces normally decay exponentially with separation distance. Here, we present experimental evidence of algebraically decaying double-layer interactions. We show that algebraic interactions arise in both strongly overlapping as well as counterion-only regimes, albeit the evidence is less clear for the former regime. In both of these cases, the disjoining pressure profile assumes an inverse square distance dependence. At small separation distances, another algebraic regime is recovered. In this regime, the pressure decays as the inverse of separation distance.

Quantification of the liquid window of deep eutectic solvents.

Deep eutectic solvents (DESs) have been considered as a new class of green solvents with tunable physical properties based on the selective combination of their individual components. As the liquid window of a DES identifies the range of feasible applications, it is essential to determine, quantify, and predict their phase behavior. Phase diagrams were measured for systems consisting of tetrapentylammonium bromide and erythritol or succinic acid. Regular solution theory is applied to quantitatively describe the liquid window of DESs. The succinic acid mixture shows a larger deviation from ideal behavior, caused by the stronger hydrogen bond forming acid groups. The interaction parameter between the two DES components in regular solution theory could be determined directly from the eutectic temperature of the mixture and this enables quantification of the degree of non-ideality of DESs.

Interactions between amphoteric surfaces with strongly overlapping double layers.

The entropic repulsion between strongly overlapping electrical double-layers from two parallel amphoteric plates is described via the Donnan equilibrium in the limit of zero electric field. The plates feature charge-regulation and the inter-plate solution is in equilibrium with a reservoir of a monovalent electrolyte solution. A finite electric potential and disjoining pressure is found at contact between the plates, due to a complete discharging of the plates. For low potentials, the decay of potential and pressure is fully governed by a characteristic length scale and the contact potential. Additionally, for large separations we find a universal inverse square decay of disjoining pressure, irrespective of the contact potential. The results of the Donnan theory show quantitative agreement with self-consistent field computations that solve the full Poisson equation.

Interfacial Tension of Phase-Separated Polydisperse Mixed Polymer Solutions.

Aqueous two-phase systems provide oil-free alternatives in the formulation of emulsions in food and other applications. Theoretical interpretation of measurements on such systems, however, is complicated by the high polydispersity of the polymers. Here, phase diagrams of demixing and interfacial tensions are determined for aqueous solutions of two large polymers present in a mass ratio of 1:1, dextran (70 kDa) and nongelling gelatin (100 kDa), with or without further addition of smaller dextran molecules (20 kDa). Both in experiments and in calculations from Scheutjens-Fleer self-consistent field lattice theory, we find that small polymers decrease the interfacial tension at equal tie-line length in the phase diagram. After identifying the partial contributions of all chemical components to the interfacial tension, we conclude that excess water at the interface is partially displaced by small polymer molecules. An interpretation in terms of the Gibbs adsorption equation provides an instructive way to describe effects of polydispersity on the interfacial tension of demixed polymer solutions.

Polyelectrolytes adsorbed at water-water interfaces.

Polyelectrolytes can show strong adsorption at water-water interfaces formed by phase separation of two polymers in aqueous solution. We demonstrate this for a model system consisting of neutral polymer A and weakly positively charged polymer B. When polyelectrolyte is added with similar chemical composition as polymer A, but charge of opposite sign as polymer B, interfacial accumulation is observed. We hypothesize this accumulation to be complexation at the water-water interface. This adsorption surprisingly persists even at high salt concentrations and has only a limited effect on the interfacial tension. Complexation of polyelectrolytes at water-water interfaces may provide a new path towards the stabilization of water-in-water emulsions.

Chemical physics of water-water interfaces.

A brief review is given on recent progress in experimental and theoretical investigations of the interface between coexisting aqueous phases of biopolymers. The experimental aspects are introduced using results obtained from a model system consisting of aqueous mixtures of nongelling gelatin and dextran. The focus is on the interfacial tension and interfacial electric potential (Donnan potential). These quantities are experimentally accessible and can be shown to be closely related.

Decreased Interfacial Tension of Demixed Aqueous Polymer Solutions due to Charge.

Electric charge at the water-water interface of demixed solutions of neutral polymer and polyelectrolyte decreases the already ultralow interfacial tension. This is demonstrated in experiments on aqueous mixtures of dextran (neutral) and nongelling fish gelatin (charged). Upon phase separation, electric charge and a potential difference develop spontaneously at the interface, decreasing the interfacial tension purely electrostatically in a way that can be accounted for quantitatively by Poisson-Boltzmann theory. Interfacial tension is a key property when it comes to manipulating the water-water interface, for instance to create novel water-in-water emulsions.

Water-in-Water Emulsions Stabilized by Nanoplates.

Ultrathin plate-like colloidal particles are effective candidates for Pickering stabilization of water-in-water emulsions, a stabilization that is complicated by the thickness and ultralow tension of the water-water interface. Plate-like particles have the advantage of blocking much of the interface while simultaneously having a low mass. Additionally, the amount of blocked interface is practically independent of the equilibrium contact angle θ at which the water-water interface contacts the nanoplates. As a result, the adsorption of nanoplates is stronger than for spheres with the same maximal cross section, except if θ = 90°.

Donnan potentials in aqueous phase-separated polymer mixtures.

A promising approach to texturize water is by the addition of mutually incompatible polymers, leading to phase separation. Here, we demonstrate that the phase stability of aqueous polymer solutions is affected not only by chemical differences between the polymers but also by their electric charge. Direct electrochemical measurements are performed of the electric potential difference between two coexisting phases in aqueous solutions of the charged protein fish gelatin (nongelling) and the uncharged polysaccharide dextran. Charge counteracts demixing because of the entropic cost of confining the counterions to one phase, resulting in a strong shift of the critical point upon an increase of the charge on one of the polymers. Upon phase separation, the charged polymer is spatially confined, and due to the Donnan effect, an interfacial electric potential is developed. A direct proportionality is found between this Donnan potential and the difference in gelatin concentration in the two phases, for which we propose a theoretical explanation. The electrostatics may provide a new handle in the development of stable water-in-water emulsions.