Complex Coacervate Phase Research Articles

Phase separation and composition of coacervates of lactoferrin and caseins

The bovine milk casein proteins (αs-casein (ACN), β-casein (BCN) and κ-casein (KCN)) form complexes with lactoferrin (LF), an anionic protein also present in bovine milk that has an important bacteriostatic function especially in infant food. LF and each casein form complexes at near neutral pH, which is in between the respective pI's (≈8.3 and ≈ 4.6). The stoichiometry of the complexes is determined by the net charge of the proteins. Optimum complexation occurs at charge neutrality and is characterized by a maximum in turbidity. Mixing LF with each casein at a ratio where charge neutrality is obtained leads to a new complex coacervate phase. The kinetics of complex formation for LF/BCN and LF/KCN is rapid and appears to occur through a nucleation and coalescence process. However, the kinetics of complex formation between LF and ACN is much slower and therefore a nucleation and growth process is proposed, based on model calculations of the turbidity. The composition of the complexes is the same as the experimental mixing ratio if mixing ratio leads to a neutral complex. On standing and light centrifugation a complex-coacervate phase is formed, which is a viscous liquid.

Characterization of fish gelatin–gum arabic complex coacervates as influenced by phase separation temperature

The rheological and structural characteristics of fish gelatin (FG)–gum arabic (GA) complex coacervate phase, separated from an aqueous mixture of 1% FG and 1% GA at pH 3.5, were investigated as influenced by phase separation temperature. Decreasing the phase separation temperature from 40 to 10°C lead to: (1) the formation of a coacervate phase with a larger volume fraction and higher biopolymer concentrations, which is more viscous, more structural resistant at low shear rates, more shear-thinning at high shear rates, and more condensed in microstructure, (2) a solid-like elastic behavior of the phase separated at 10°C at a high oscillatory frequency, (3) the increase in gelling and melting temperatures of the coacervate phase (3.7−3.9°C and 6.2−6.9°C, respectively), (4) the formation of a more rigid and thermo-stable coacervate gel. The coacervate phase is regarded as a homogeneously networked biopolymer matrix dispersed with water vacuoles and its gel as a weak physical gel reinforced by FG–GA attractive electrostatic interactions.

Complex coacervates of lactotransferrin and β-lactoglobulin

HypothesisOppositely charged proteins should interact and form complex coacervates or precipitates at the correct mixing ratios and under defined pH conditions. ExperimentsThe cationic protein lactotransferrin (LF) was mixed with the anionic protein β-lactoglobulin (B-Lg) at a range of pH and mixing ratios. Complexation was monitored through turbidity and zeta potential measurements. FindingsComplexation between LF and B-Lg did occur and complex coacervates were formed. This behaviour for globular proteins is rare. The charge ratio’s of LF:B-Lg varies with pH due to changing (de) protonation of the proteins. Nevertheless we found that the complexes have a constant stoichiometry LF:B-Lg=1:3 at all pH’s, due to charge regularization. At the turbidity maximum the zeta potential of complexes is close to zero, indicating charge neutrality; this is required when the complexes form a new concentrated liquid phase, as this must be electrically neutral. Complexes were formed in pH region 5–7.3. On addition of salt (NaCl) complexation is diminished and disappears at a salt concentration of about 100mMol. The coacervate phase has a very viscous consistency. If we consider the proteins as colloidal particles then the formed complex coacervate phase may have a structure that resembles a molten salt comparable to, for example, AlCl3.

Interfacial Tension of Polyelectrolyte Complex Coacervate Phases.

We consider polyelectrolyte solutions which, under suitable conditions, phase separate into a liquid-like coacervate phase and a coexisting supernatant phase that exhibit an extremely low interfacial tension. Such interfacial tension provides the basis for most coacervate-based applications, but little is known about it, including its dependence on molecular weight, charge density, and salt concentration. By combining a Debye-Hückel treatment for electrostatic interactions with the Cahn-Hilliard theory, we derive explicit expressions for this interfacial tension. In the absence of added salts, we find that the interfacial tension scales as N-3/2(η/ηc-1)3/2 near the critical point of the demixing transition, and that it scales as η1/2 far away from it, where N is the chain length and η measures the electrostatic interaction strength as a function of temperature, dielectric constant, and charge density of the polyelectrolytes. For the case with added salts, we find that the interfacial tension scales with the salt concentration ψ as N-1/4(1-ψ/ψc)3/2 near the critical salt concentration ψc. Our predictions are shown to be in quantitative agreement with experiments and provide a means to design new materials based on polyelectrolyte complexation.

Fluidity and water in nanoscale domains define coacervate hydrogels

Coacervate-based hydrogels, formed in aqueous solution by simple mixing of two oppositely charged ABA block copolyelectrolytes represent a new and versatile approach to the design of bio-inspired gelators. While coacervate-based hydrogels provide high tunability of a range of desirable properties, little is understood about the molecular-level makeup of the nanometer-scale domains. Small angle neutron scattering was employed to quantify the effective polymer density and water content of each domain. Further, electron paramagnetic resonance and Overhauser dynamic nuclear polarization of block-specific spin labels elucidate domain-specific, local, polymer and water dynamics. This unique combination of techniques reveals that the charged A blocks segregate into spherical domains with a radius of 8 nm, and are dispersed in a continuous matrix of water soluble, PEO B blocks. The edges of the spherical A block domains are found to be soft and diffuse, and the B block matrix exhibits higher water and polymer dynamics than the A block domains. The selective measurement of the local water and polymer dynamics shows a viscous and dense, but fluidic environment in the spherical A block domains, thus permitting the designation as a complex coacervate phase. Further, the physical properties of the analogous homopolymers mixed at equal composition to that of the triblock copolyelectrolytes leads to the conclusion that “the whole is greater than the sum of its parts”: nanometer scale complex coacervates only form when the two charged A blocks are covalently linked by a PEO midblock that serves as an intrinsic osmolyte.

Interrelationship between the zeta potential and viscoelastic properties in coacervates complexes

The formation of the complex coacervate (CC) phases between gum Arabic (GA) and low molecular weight chitosan (Ch) and the interrelationship between the zeta-potential and viscoelastic properties of the coacervate phase were investigated. The maximum charge difference of biopolymers stock dispersion was displayed in a range of pH between 4.0 and 5.5. Titration experiment between the oppositely charged biopolymers showed that the isoelectric point was found at a biopolymers mass ratio (R[GA:Ch]) of R[5.5:1]. Turbidity, size and ζ-potential of the soluble complexes (SC) showed an interrelation with the complex coacervate yield (CCY). Higher CCY values (82.2–88.1%) were obtained in the range from R[3:1] to R[5.5:1]. Change the R[GA:Ch] in dispersion, make possible to produce CC's phases exhibiting cationic (R[1:1] and R[3:1]), neutral (R[5.5:1]) or anionic (R[9:1] and R[7:1]) charged. All CC's exhibited liquid-viscoelastic behavior at lower frequencies and a crossover between G″ and G′ at higher frequencies.

Linear Viscoelasticity of Polyelectrolyte Complex Coacervates

Two flexible, oppositely charged polymers can form liquid-like complex coacervate phases with rich but poorly understood viscoelastic properties. They serve as an experimental model system for many biological and man-made materials made from oppositely charged macromolecules. We use rheology to systematically study the viscoelastic properties as a function of salt concentration, chain length, chain length matching, and mixing stoichiometry of model complex coacervates of poly(N,N-dimethylaminoethyl methacrylate), PDMAEMA, and poly(acrylic acid), PAA. The dynamics of making and breaking ionic bonds between the oppositely charged chains underlie all linear viscoelastic properties of the complex coacervates. We treat (clusters of) ionic bonds as sticky points and find that there is a remarkable resemblance between the relaxation spectra of these complex coacervates and the classical sticky Rouse model for single polymer systems. Salt affects all relaxation processes in the same way, giving rise to a widely a...

Polyplexes of Poly(methylaminophosphazene): Energetics of DNA Melting

The interaction of DNA with a synthetic biocompatible and biodegradable cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP·HCl), was investigated by high-sensitivity differential scanning calorimetry under conditions of strong and weak electrostatic interactions of the macroions. Thermodynamic parameters of the DNA double-helix melting were determined as a function of pH and the PMAP·HCl/DNA weight ratio. PMAP·HCL was shown to reveal two functions with respect to DNA: the polyelectrolyte function and the donor-acceptor one. The first function stabilizes the helical conformation of DNA, and the second one destabilizes it. The stabilizing effect of PMAP·HCl is of entropic origin, related to a displacement of mobile counterions from the DNA's nearest surroundings by the poly(methylaminophosphazene) charged groups. The donor-acceptor function of poly(methylaminophosphazene) dominates when its electrostatic interaction with DNA is either saturated (in the complex coacervate phase at high poly(methylaminophosphazene) concentrations) or completely suppressed (in a salt medium when the polycation carries a small charge). Under these conditions, poly(methylaminophosphazene) destabilizes DNA. It preferentially binds to the DNA coil form likely via the formation of multiple labile hydrogen bonds with the donor-acceptor groups of DNA.

Interfacial tension between a complex coacervate phase and its coexisting aqueous phase

Complex coacervation is the associative phase separation in a solution of positively and negatively charged macroions. Despite the widespread use of coacervation in e.g. micellar assemblies (complex coacervate core micelles), drug carriers and thin films, there is virtually no experimental data on the interfacial tension between such coacervate phases (polyelectrolyte complexes) and their coexisting aqueous phases or on the influence of salt thereon. In this paper we use colloidal probe AFM measurements of capillary adhesion forces to obtain the interfacial tension between a complex coacervate phase of two polyelectrolytes with high charge density and its coexisting aqueous phase. We find that the interfacial tension is of order 100 µN/m, decreases with increasing salt concentration and vanishes at the critical point. Interestingly, we find that the critical scaling exponent for the interfacial tension found in segregative demixing also applies here.

Local Water Dynamics in Coacervated Polyelectrolytes Monitored through Dynamic Nuclear Polarization-Enhanced 1H NMR

We present the first study of quantifying the diffusion coefficient of interfacial water on polyelectrolyte surfaces of systems fully dispersed in bulk water under ambient conditions. Such measurements were made possible through the implementation of a recently introduced Dynamic Nuclear Polarization (DNP) technique to selectively amplify the nuclear magnetic resonance (NMR) signal of hydration water that is interacting with specifically located spin labels on polyelectrolyte surfaces. The merit of this novel capability is demonstrated in this report through the measurement of solvent microvisosity on the surface of two types of oppositely charged polyelectrolytes, when freely dissolved versus when complexed to form a liquid-liquid colloidal phase called complex coacervates. These complex coacervates were formed through electrostatic complexation between the imidazole-based cationic homopolymer poly(N-vinylimidazole) (PVIm), and anionic polypeptide polyaspartate (PAsp) in the pH range of 4.5 - 6.0, under which conditions the coacervate droplets are highly fluidic yet densely packed with polyelectrolytes. We also investigated the rotational diffusion coefficients of the spin labels covalently bound to the polyelectrolyte chains for both PVIm and PAsp, showing a 5 fold change in the rotational correlation time as well as anisotropy parameter upon coacervation, which represents a surprisingly small decrease given the high polymer concentration inside the dense microdroplets. For both DNP and ESR experiments, the polymers were covalently tagged with stable nitroxide radical spin labels (∼1 wt %) to probe the local solvent and polymer segment dynamics. We found that the surface water diffusion coefficients near uncomplexed PVIm and PAsp at pH 8 differ, and are around D∼1.3×10(-9)m(2) / s. In contrast, inside the complex coacervate phase, the water diffusion coefficient in the immediate vicinity of either polyelectrolyte was D∼ 0.25×10(-9)m(2) / s, which is about an order of magnitude smaller than the bulk water self diffusion coefficient, and yet orders of magnitude greater than that of associated, bound, hydration water. This observation suggests the existence of measurable water inside complex coacervates with relatively high diffusion and exchange dynamics, implying that water moves in nanometer-scale pore spaces as opposed to being structurally bound or even absent. We infer from our observation that the PVIm and PAsp chains are undergoing roughly pairwise association, so that largely charge neutralized species compose the concentrated, yet fluidic and partially hydrated coacervate cores.

Phase behavior of gelatin in the presence of pectin in water-acid medium

Phase separation of alkaline gelatin in water-acid solutions in the presence of low etherified pectin (ED 38%) were investigated. The effects of the pectin weight fraction in pectin/gelatin mixture (qo) as well as two conditions of complex formation, namely, mixing of the binary biopolymer-solvent systems at pH 3.5 (‘mixing conditions’), or preparation of the ternary gelatin-pectin-water systems at pH 7.5 and their subsequent acidification up to pH 3.5 (‘titration conditions’), on phase equilibrium and macrostructure of the concentrated complex phase were established using phase analysis, and optical microscopy. At qo 0.5 unusual three phase separation took place in the ‘mixing conditions’, leading to formation of supernatant (phase 1), complex coacervate (phase 2) and concentrated pectin solution (phase 3). Possible mechanism of such phenomenon was discussed in term of segregative and aggregative phase separations.