Studies of the stability of water-soluble polypeptoid helices and investigation of synthetic, biomimetic substrates for the development of a thermally triggered, enzymatically crosslinked hydrogel for biomedical applications

Due to the unique 3D structures of proteins, these biopolymers are able to perform a myriad of vital functions and activities in vivo. Peptidomimetic oligomers are being synthesized to mimic the structure and function of natural peptides. We have examined the stability of secondary structure of a poly-N-substituted glycine (peptoid) and developed synthetic substrates for transglutaminase enzymes. We synthesized an amphipathic, helical, 36 residue peptoid to study the stability of peptoid secondary structure using circular dichroism. We saw no significant dependence of helical structure on concentration, solvent, or temperature. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role, of steric forces in their structural stabilization. The structured polypeptoids studied here have potential as robust mimics of helical polypeptides of therapeutic interest.; The ability of transglutaminases to crosslink peptidomimetic substrates was also investigated. There is a medical need for robust, biocompatible hydrogels that can be rapidly crosslinked in situ, for application as surgical adhesives, bone-inductive materials, or for drug delivery. We have taken an enzymatic approach to the creation of a novel gelation system that fits these requirements, utilizing transglutaminase enzymes, thermo-responsive liposomes, and a biomimetic enzyme substrate based on a peptide-polymer conjugate. At room temperature, the hydrogel system is a solution. Upon heating to 37°C, the calcium-loaded liposomes release calcium that activates Factor XIII in the presence of thrombin, producing a gel within 9 minutes. Rheological studies demonstrated that the hydrogel behaves as a robust, elastic solid, while scanning electron microscopy studies revealed that the hydrogel has a very dense morphology overall. We also investigated the ability of transglutaminases to crosslink non-natural, peptoid-based substrates. The activity of five lysine-containing peptoid substrates and two glutamine-containing peptoid substrates with proteinogenic side chains were compared to their peptide analogs. Lysine-containing peptoid substrates were crosslinked by the transglutaminase but at a much lower rate, producing at most 28% of the crosslinked product that its peptide counterpart produced. Of the two glutamine-containing peptoid substrates investigated, one did not show any crosslinked product formation, while the other was insoluble in aqueous solution.