Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles.

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, poly(oligoethylene glycol methacrylate-block-quaternized 4-vinylpyridine), POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte complexes to deliver anionic proteins intracellularly. Using this model system, we have identified requirements for the formation of PEC micelles that are stable at physiological ionic strength and that smaller protein-polyelectrolyte complexes effectively deliver proteins to Jurkat cells.

Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress.

Protein-containing polyelectrolyte complexes (PECs) are a diverse class of materials, composed of two or more oppositely charged polyelectrolytes that condense and phase separate near overall charge neutrality. Such phase-separation can take on a variety of morphologies from macrophase separated liquid condensates, to solid precipitates, to monodispersed spherical micelles. In this review, we present an overview of recent advances in protein-containing PECs, with an overall goal of defining relevant design parameters for macro- and microphase separated PECs. For both classes of PECs, the influence of protein characteristics, such as surface charge and patchiness, co-polyelectrolyte characteristics, such as charge density and structure, and overall solution characteristics, such as salt concentration and pH, are considered. After overall design features are established, potential applications in food processing, biosensing, drug delivery, and protein purification are discussed and recent characterization techniques for protein-containing PECs are highlighted.

Ionic polypeptide tags for protein phase separation.

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. The protein design factors that govern complex coacervation under varying conditions are still relatively unexplored. Recent work indicates that proteins with an intrinsically disordered region, a higher net charge, or a patch of charged residues are more likely to undergo a phase transition. Based on these design parameters, polyionic coacervation tags were designed and assessed for their ability to promote protein complex coacervation with oppositely charged polyelectrolytes. The phase behavior of a panel of engineered proteins was evaluated with the strong polycation poly(4-vinyl N-methyl pyridinium iodide). Proteins containing the ionic tags formed liquid coacervate droplets, while isotropically charged protein variants formed solid precipitates. The ionic tags also promoted phase separation at higher salt concentrations than an isotropic distribution of charge on the protein surface. The salt dependence of the protein complex coacervation could be predicted independently for tagged or isotropic variants by the ratio of negative-to-positive residues on the proteins and universally by calculating the distance between like charges. The addition of just a six residue polyionic tag generated a globular protein capable of liquid-liquid phase separation at physiological pH and ionic strength. This model system has provided the initial demonstration that short, ionic polypeptide sequences (6-18 amino acids) can drive the liquid-liquid phase separation of globular proteins.