Covalently crosslinked coacervates: immobilization and stabilization of proteins with enhanced enzymatic activity

Coacervates represent models for membrane-free protocells and thus provide a simple route to synthetic cellular-like systems that provide selective encapsulation of solutes. Here, we demonstrate a simple and versatile post-coacervation crosslink method using the thiol-ene click reaction in aqueous media to prepare covalently crosslinked coacervates. The crosslinking of the coacervate enables stability at extreme pH where the uncrosslinked coacervate fully disassembles. The crosslinking also enhances the hydrophobicity within the coacervate environment to increase the encapsulation efficiency of bovine serum albumin (BSA), as compared to the uncrosslinked coacervate. Additionally, the crosslinked coacervate increases the stabilization of BSA at low pH. These crosslinked coacervates can act as carriers for enzymes. The enzymatic activity of alkaline phosphatase (ALP) is enhanced within the crosslinked coacervate compared to the ALP in aqueous solution. The post-coacervation crosslink approach allows the utilization of coacervates for encapsulation of biologicals under conditions where the coacervate would generally disassemble. We demonstrate that these crosslinked coacervates enable the protection of encapsulated protein against denaturation at extreme pH and enhance the enzymatic activity with encapsulation. This click approach to stabilization of coacervates should be broadly applicable to other systems for a variety of biologics and environmentally sensitive molecules.