Dimitrios Priftis

Phase behaviour and complex coacervation of aqueous polypeptide solutions

Mixing of oppositely charged polyelectrolytes in aqueous solutions may result in the formation of polyelectrolyte complexes (PEC). Phase separation and complex coacervation of polypeptides are investigated in this study. Polypeptides have identical backbones and differ only in their charged side groups, making them attractive model systems for this work. All experiments are conducted using equal chain lengths of polycation and polyanion in order to isolate and highlight effects of the interactions of the charged groups during complexation. Complex coacervation is strongly affected by the polypeptide mixing ratio (stoichiometry), ionic strength (salt concentration), total polymer concentration, pH, and temperature. To examine the effect of these parameters on complex formation we use sample turbidity as an indicator, and optical microscopy to discriminate between coacervate and precipitate. We establish phase diagrams as a function of polybase content and salt concentration using the critical salt concentrations required to reach the coacervate to solution boundary. Additionally, we examine the effect of molecular weight on the complex formation for the P(L-Lysine) (PLys)/P(L-Glutamic acid) (PGlu) system and establish a phase diagram. By determining the water content of the coacervate phase under various conditions we find that the salt content and stoichiometry of the mixed polyelectrolytes have a significant effect on the coacervate composition.

Thermodynamic characterization of polypeptide complex coacervation.

The interactions between a series of oppositely charged polypeptide pairs are probed using isothermal titration calorimetry (ITC) in combination with turbidity measurements and optical microscopy. Polypeptide complex coacervation is described as a sequence of two distinct binding steps using an empirical extension of a simple ITC binding model. The first step consists of the formation of soluble complexes from oppositely charged polypeptides (ion pairing), which in turn aggregate into insoluble interpolymer complexes in the second step (complex coacervation). Polypeptides have identical backbones and differ only in their charged side groups, making them attractive model systems for this work. The poly(l-ornithine hydrobromide) (PO)/poly(l-glutamic acid sodium salt) (PGlu) system is used to examine the effects of parameters such as the salt concentration, pH, temperature, degree of polymerization, and total polymer concentration on the thermodynamic characteristics of complexation. Complex coacervation in all probed systems is found to be endothermic, essentially an entropy-driven processes. Increasing the screening effect of the salt on the polyelectrolyte charges diminishes their propensity to interact, leading to a decrease in the observed energy change and coacervate quantity. The pH plays an important role in complex formation through its effect on the degree of ionization of the functional groups. Plotting the change in enthalpy with temperature allows the calculation of the heat capacity change (ΔC(p)) for the PO/PGlu interactions. Finally, ITC revealed that complex coacervation is promoted when higher total polymer concentrations or polypeptide chain lengths are used.

Chirality-selected phase behaviour in ionic polypeptide complexes

Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

Interfacial Energy of Polypeptide Complex Coacervates Measured via Capillary Adhesion

A systematic study of the interfacial energy (γ) of polypeptide complex coacervates in aqueous solution was performed using a surface forces apparatus (SFA). Poly(L-lysine hydrochloride) (PLys) and poly(L-glutamic acid sodium salt) (PGA) were investigated as a model pair of oppositely charged weak polyelectrolytes. These two synthetic polypeptides of natural amino acids have identical backbones and differ only in their charged side groups. All experiments were conducted using equal chain lengths of PLys and PGA in order to isolate and highlight effects of the interactions of the charged groups during complexation. Complex coacervates resulted from mixing very dilute aqueous salt solutions of PLys and PGA. Two phases in equilibrium evolved under the conditions used: a dense polymer-rich coacervate phase and a dilute polymer-deficient aqueous phase. Capillary adhesion, associated with a coacervate meniscus bridge between two mica surfaces, was measured upon the separation of the two surfaces. This adhesion enabled the determination of the γ at the aqueous/coacervate phase interface. Important experimental factors affecting these measurements were varied and are discussed, including the compression force (1.3-35.9 mN/m) and separation speed (2.4-33.2 nm/s). Physical parameters of the system, such as the salt concentration (100-600 mM) and polypeptide chain length (N = 30, 200, and 400) were also studied. The γ of these polypeptide coacervates was separately found to decrease with both increasing salt concentration and decreasing polypeptide chain length. In most of the above cases, γ measurements were found to be very low, <1 mJ/m(2). Biocompatible complex coacervates with low γ have a strong potential for applications in surface coatings, adhesives, and the encapsulation of a wide range of materials.

Complex coacervation of poly(ethylene-imine)/polypeptide aqueous solutions: thermodynamic and rheological characterization.

This study is aimed at understanding the complex coacervation of two systems: branched poly(ethyleneimine) with linear poly(D,L-glutamic acid) or poly(D,L-aspartic acid), and identify differences and similarities with previously reported systems. Three different techniques (turbidity, isothermal titration microcalorimetry-ITC and rheology) were used in a comprehensive study of coacervation. Sample turbidity was used to show how various parameters (salt, stoichiometry, pH, temperature) affect complex coacervation. Complex coacervation decreases with increase in salt and coacervate formation is maximum when a 31:69 mol% acid:base ratio is used. Rare in literature phase diagrams revealed that coacervates are formed over a wide range of acid:base ratios (15-88 mol% NH3(+) groups), significantly broader compared to other systems. ITC was used for the thermodynamic characterization of the complexation between the polyelectrolytes, and showed that complex coacervation is entropy-driven (from the release of counter-ions) and enthalpically unfavored process. Composition and viscoelastic properties of the complex coacervates were examined gravimetrically and through rheology. Coacervate water content depends on the salt concentration and the stoichiometry. Coacervates exhibit a viscoelastic behavior that is dependent on the salt concentration. Master curves that can predict behavior at a wide range of time scales, not accessible by conventional rheological measurements, were created.

Self‐Assembly of α‐Helical Polypeptides Driven by Complex Coacervation

Reported is the ability of α-helical polypeptides to self-assemble with oppositely-charged polypeptides to form liquid complexes while maintaining their α-helical secondary structure. Coupling the α-helical polypeptide to a neutral, hydrophilic polymer and subsequent complexation enables the formation of nanoscale coacervate-core micelles. While previous reports on polypeptide complexation demonstrated a critical dependence of the nature of the complex (liquid versus solid) on chirality, the α-helical structure of the positively charged polypeptide prevents the formation of β-sheets, which would otherwise drive the assembly into a solid state, thereby, enabling coacervate formation between two chiral components. The higher charge density of the assembly, a result of the folding of the α-helical polypeptide, provides enhanced resistance to salts known to inhibit polypeptide complexation. The unique combination of properties of these materials can enhance the known potential of fluid polypeptide complexes for delivery of biologically relevant molecules.

Correction: Phase behaviour and complex coacervation of aqueous polypeptide solutions

Correction for ‘Phase behaviour and complex coacervation of aqueous polypeptide solutions’ by Dimitrios Priftis et al., Soft Matter, 2012, 8, 9396–9405.