Thermodynamic Characterization of Polypeptide Complex Coacervation

Abstract

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.

Linear viscoelasticity of complex coacervates

“…Characterization of coacervate phase behavior is typically performed using methods that take advantage of the light scattered by these small droplets in solution, such as turbidity and/or light scattering. While rheology can be used to identify the advent of complex formation based on an increase in viscosity [124], optical methods have been more widely utilized because they are amenable for high-throughput analysis to quantify the impact of variables such as the charge stoichiometry of the mixture, the ionic strength and pH of the solution, the total concentration of macro-ions, the charge density, and for samples containing polyelectrolytes, the polymer molecular weight [1][2][3]17,69,.…”

“…The self-assembly of these materials is driven by entropy, where the initial electrostatic attraction between oppositely-charged macro-ions results in the release of small, bound counter-ions and the restructuring of water molecules [1][2][3][4]. Complex coacervates have a long history of use in the food [5][6][7][8][9][10][11][12][13] and personal care [14,15] industries, and have found increasing utility as a platform for drug and gene delivery [1][2][3][4], as well as underwater adhesives [5][6][7][8][9][10][11][12][13][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62]. Coacervation has also recently been implicated in the formation of various biological assemblies [1,[14][15][16]55,…”

“…Coacervation has also recently been implicated in the formation of various biological assemblies [1,[14][15][16]55,[63][64][65][66][67][68][69][70]. Across nearly all of these applications, the vast majority of studies have focused on understanding and characterizing the equilibrium phase behavior of these materials as a function of parameters such as the chemistry of the charged species, the charge stoichiometry of the system, ionic strength, and pH [1][2][3]17,69,. However, these types of equilibrium characterizations do not provide sufficient insight into the dynamic behavior of coacervates.…”

PRISM-Based Theory of Complex Coacervation: Excluded Volume versus Chain Correlation

“…2,[31][32][33] The novelty of these materials is their extreme stability (yet responsiveness) with respect to environmental ionic conditions, even at high salt concentrations, as well as their reversible assembly behavior. [34][35][36][37][38] Complex coacervates are used for microencapsulation, drug delivery, 3,19 biomaterials, [39][40][41] and underwater adhesives, 2,31,32 where their self assembly and functionality relies 19,[23][24][25][26] on the relationship between charged species. Thus, the molecular features of these charged species is crucial to designing function.…”

“…1 Polymeric complex coacervates demonstrate a rich phenomenology that is a function of the presence of salt as well as the nature of the charged polyions, 1,32,[34][35][36][37][38][47][48][49][50] with a vast parameter space that spans the molecular features of all four components. Experimentalists have begun to systematically explore this space, looking at effects such as chain solubility and length, ion identity, and valency.…”