Trehalose Glycopolymers and Hydrogels for Enhancing Protein Stability

Author(s): Lee, Juneyoung | Advisor(s): Maynard, Heather D | Abstract: Proteins are widely used in broad regions of interest from laboratories to industries and from reaction catalysts to therapeutic agents. However, the instability of proteins toward various environmental stressors both in storage and in vivo is a barrier to their general use. In this dissertation, development of trehalose glycopolymers and hydrogels to enhance protein stability and their pharmacokinetic properties is described. Traditional polymers used to prevent degradation by thermal stress and lyophilization are shown in Chapter 1. Chapter 2 describes the synthesis of three trehalose-based monomers and polymers. Trehalose glycopolymers were synthesized using free radical polymerization with trehalose moieties as side chains. The resulting polymers were then used as excipients to stabilize horseradish peroxidase, glucose oxidase, and beta-galactosidase during heating and lyophilization processes. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. In Chapter 3, the conjugation of trehalose polymers to therapeutic proteins, including insulin and granulocyte colony-stimulating factor, to enhance stability and improve pharmacokinetic properties is described. First, the insulin structure was monitored by native gel before and after heating an insulin solution to 90 i?½C for 1 h. The structure of insulin was maintained when trehalose polymers were added as additives whereas insulin alone or with trehalose as an additive completely or partially lost its original structure. To prepare polymers with protein reactive end-groups, ketone and benzaldehyde end-group functionalized chain transfer agents were synthesized and used to mediate reversible addition-fragmentation chain transfer polymerization of trehalose monomers. The prepared polymers were covalently conjugated to insulin and granulocyte colony-stimulating factor through reductive amination. Pharmacokinetic studies using mice showed a dramatic increase in the half-life of insulin-trehalose polymer conjugates compared to native insulin and the effect was similar to that of insulin-poly(ethylene glycol) conjugates. Also, the synthesis of a trehalose polymer with aminooxy end-group that could form an oxime bond and modification of cell surface with ketone functional group are presented. Chapter 4 demonstrates the preparation of trehalose and hyaluronic acid-based delivery agents. There are four different approaches discussed to enable stabilization and delivery of proteins. With the described technologies, the protein may be delivered passively or by triggered release. In Chapter 5, we report the use of a trehalose polymer as a new resist that allows direct electron beam lithography writing of multiple proteins. First, trehalose glycopolymer was shown to effectively crosslink to surfaces as negative resists. Also, it was observed that proteins were stabilized during electron beam exposure and vacuum conditions when the trehalose glycopolymer was added into the protein solution. Using this technique we could directly pattern multiple proteins at the micrometer and nanometer scale without additional steps, such as preparing protein-reactive polymer or conjugating proteins to polymer patterns. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins were successfully generated.