Introduction: Therapeutic proteins are challenging to transport and store and the majority need to be refrigerated or frozen. Proteins exposed to these stressors often lose activity. This can be harmful or even fatal for patients that take the medicines. Thus polymers and hydrogels that are capable of stabilizing biomolecules at room temperature are of significant interest. The disaccharide sugar, trehalose, is known to stabilize proteins to a variety of environments including to elevated temperatures and desiccation. We hypothesized that by preparing polymers with side chain trehalose units, the resulting materials would also stabilize proteins. This talk will focus on linear polymer conjugates and hydrogels with trehalose as a component for protein stabilization. Results and Discussion: A library of polymers with polystyrene or polymethacrylate backbones and trehalose side chains was prepared by free radical polymerization. Therapeutic proteins such as insulin were tested under stress conditions such as heat with and without added polymer. The polymers that performed optimally in this exercise were utilized to prepare conjugates with the therapeutic proteins. This was accomplished by synthesizing the polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization utilizing functional chain transfer agents (CTA) so that the resulting polymers contained end groups reactive to amino acid side chains on proteins. The polymers were then conjugated to the proteins and the resulting conjugates purified by fast protein liquid chromatography. Pharmacokinetics in mice, in vitro and in vivo bioactivity, and stability to elevated temperatures were evaluated. The results showed that the trehalose polymers were able to (a) enhance the plasma half life similar to gold standard poly(ethylene glycol) (PEG), (b) remain bioactive similar to the analogous PEGylated proteins, and (c) stabilize the proteins to elevated temperatures. They were also non-toxic in vitro and in vivo. Discussion of this research will encompass the first part of the talk. Hydrogels were also prepared by either free radical polymerization of the trehalose monomers and trehalose crosslinkers or by specific condensation reactions between the trehalose polymers and boronic acid end-functionalized PEGs. Both cases resulted in gels that could incorporate and stabilize proteins such as insulin to thermal stress. The gels released the proteins by passive release or by triggered release by glucose, respectively. This research will be discussed in the second part of this talk. Conclusions: Trehalose materials, also called PolyProtek, are excellent protein stabilizers. Make in a linear form, the polymers form therapeutic protein conjugates that are bioactive and stable to temperatures and in vivo. As a hydrogel the materials can stabilize prior to triggered release of proteins. Thus, trehalose materials are promising for therapeutic applications.
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