Transfer Matrix Model of pH Effects in Polymeric Complex Coacervation.

Oppositely charged polyelectrolytes can undergo an associative phase separation, in a process known as polymeric complex coacervation. This phenomenon is driven by the electrostatic attraction between polyanion and polycation species, leading to the formation of a polymer-dense coacervate phase and a coexisting polymer-dilute supernatant phase. There has been significant recent interest in the physical origin and features of coacervation; yet notably, experiments often use weak polyelectrolytes the charge state of which depends on solution pH, while theoretical or computational efforts typically assume strong polyelectrolytes that remain fully charged. There have been only a few efforts to address this limitation, and thus there has been little exploration of how pH can affect complex coacervation. In this paper, we modify a transfer matrix theory of coacervation to account for acid-base equilibria, taking advantage of its ability to directly account for some local ion correlations that will affect monomer charging. We show that coacervation can stabilize the charged state of a weak polyelectrolyte via the proximity of oppositely charged monomers, and can lead to asymmetric phase diagrams where the positively and negatively charged polyelectrolytes exhibit different behaviors near the pKa of either chain. Specifically, there is a partitioning of one of the salt species to a coacervate to maintain electroneutrality when one of the polyelectrolytes is only partially charged. This results in the depletion of the same salt species in the supernatant, and overall can suppress phase separation. We also demonstrate that, when one of the species is only partially charged, mixtures that are off-stoichiometric in volume fraction but stoichiometric in charge exhibit the greatest propensity to form coacervate phases.

Incorporation of proteins into complex coacervates.

Complex coacervates have found a renewed interest in the past few decades in various fields such as food and personal care products, membraneless cellular compartments, the origin of life, and, most notably, as a mode of transport and stabilization of drugs. Here, we describe general methods for characterizing the phase behavior of complex coacervates and quantifying the incorporation of proteins into these phase separated materials.

Thermostabilization of viruses via complex coacervation.

Widespread vaccine coverage for viral diseases could save the lives of millions of people each year. For viral vaccines to be effective, they must be transported and stored in a narrow temperature range of 2-8 °C. If temperatures are not maintained, the vaccine may lose its potency and would no longer be effective in fighting disease; this is called the cold storage problem. Finding a way to thermally stabilize a virus and end the need to transport and store vaccines at refrigeration temperatures will increase access to life-saving vaccines. We explore the use of polymer-rich complex coacervates to stabilize viruses. We have developed a method of encapsulating virus particles in liquid complex coacervates that relies on the electrostatic interaction of viruses with polypeptides. In particular, we tested the incorporation of two model viruses; a non-enveloped porcine parvovirus (PPV) and an enveloped bovine viral diarrhea virus (BVDV) into coacervates formed from poly(lysine) and poly(glutamate). We identified optimal conditions (i.e., the relative amount of the two polypeptides) for virus encapsulation, and trends in this composition matched differences in the isoelectric point of the two viruses. Furthermore, we were able to achieve a ∼103-104-fold concentration of virus into the coacervate phase, such that the level of virus remaining in the bulk solution approached our limit of detection. Lastly, we demonstrated a significant enhancement of the stability of non-enveloped PPV during an accelerated aging study at 60 °C over the course of a week. Our results suggest the potential for using coacervation to aid in the purification and formulation of both enveloped and non-enveloped viruses, and that coacervate-based formulations could help limit the need for cold storage throughout the transportation and storage of vaccines based on non-enveloped viruses.

The benefit of poor mixing: kinetics of coacervation.

Complex coacervation has become a prominent area of research in the fields of food science, personal care, drug stabilization, and more. However, little has been reported on the kinetics of assembly of coacervation itself. Here, we describe a simple, low-cost way of looking at the kinetics of coacervation by creating poorly mixed samples. In particular, we examine how polymer chain length, the patterning and symmetry of charges on the oppositely charged polyelectrolytes, and the presence of salt and a zwitterionic buffer affect the kinetics of complex coacervation. Our results suggest an interesting relationship between the time for equilibration and the order of addition of polymers with asymmetric patterns of charge. Furthermore, we demonstrated that increasing polymer chain length resulted in a non-monotonic trend in the sample equilibration times as a result of opposing factors such as excluded volume and diffusion. We also observed differences in the rate of sample equilibration based on the presence of a neutral, zwitterionic buffer, as well as the presence and identity of added salt, consistent with previous reports of salt-specific effects on the rheology of complex coacervates. While not a replacement for more advanced characterization strategies, this turbidity-based method could serve as a screening tool to identify interesting and unique phenomena for further study.

Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us.

Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted.

Correction: Design rules for encapsulating proteins into complex coacervates.

Correction for 'Design rules for encapsulating proteins into complex coacervates' by Whitney C. Blocher McTigue et al., Soft Matter, 2019, 15, 3089-3103.

Design rules for encapsulating proteins into complex coacervates.

We investigated the encapsulation of the model proteins bovine serum albumin (BSA), human hemoglobin (Hb), and hen egg white lysozyme (HEWL) into two-polymer complex coacervates as a function of polymer and solution conditions. Electrostatic parameters such as pH, protein net charge, salt concentration, and polymer charge density can be used to modulate protein uptake. While the use of a two-polymer coacervation system enables the encapsulation of weakly charged proteins that would otherwise require chemical modification to facilitate electrostatic complexation, we observed significantly higher uptake for proteins whose structure includes a cluster of like-charged residues on the protein surface. In addition to enhancing uptake, the presence of a charge patch also increased the sensitivity of the system to modulation by other parameters, including the length of the complexing polymers. Lastly, our results suggest that the distribution of charge on a protein surface may lead to different scaling behaviour for both the encapsulation efficiency and partition coefficient as a function of the absolute difference between the protein isoelectric point and the solution pH. These results provide insight into possible biophysical mechanisms whereby cells can control the uptake of proteins into coacervate-like granules, and suggest future utility in applications ranging from medicine and sensing to remediation and biocatalysis.