Peptides and proteins have long been used as therapeutics to treat disease. However, there are several challenges in effectively delivering these biologics to the body, including degradation by proteolytic enzymes, short circulation life, rapid kidney clearance, and tendency to produce an immune response. One of the traditionally used methods for overcoming these issues is PEGylation. Although PEGylation has been shown to effectively stabilize the protein, it can result in a significant loss in enzyme activity and be difficult to separate and scale-up. Recently, controlled radical polymerization (CRP) techniques have been used to yield the same stabilization effects while overcoming some of the challenges that come along with PEGylation. CRP techniques such as atom-transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT) have been shown to improve protein stability while retaining most of the enzyme activity and giving control over the type of monomer used and size of polymer created. RAFT has some advantages over the other CRP techniques because it does not require a transition metal catalyst and RAFT has excellent tolerance to functional groups. With RAFT, both a grafting-to and grafting-from technique can be used to attach a polymer to a protein or grow a polymer off a protein, respectively. The grafting-from method makes purifying the protein-polymer conjugates easier, so the first method attempted involved conjugating chain transfer agents (CTAs) to the lysine residues of hen egg white lysozyme, followed by RAFT polymerization to grow PEG acrylate chains from the CTA modified protein. When the CTA was attached to the lysine residues, the protein became water insoluble with increasing attachments. To improve the solubility of this RAFT agent a short macro-CTA of acrylamide was grown in solvent first, which was subsequently conjugated to the protein in a buffer solution. The macro-CTA was chain extended with functional monomers to create a library of protein-polymer conjugates with different chain lengths and chemical functionalities. These protein-CTA and protein-polymer conjugates were analyzed to determine the number CTA of attachments, polymer length, enzyme activity retention, and stability.

Tailoring the activity of lysozyme by combining grafting-from and grafting-To RAFT polymerization

249th National Meeting (2015)

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