Poster P3 - Physically entangled hydrogels derived from bioorthogonally polymerized coiled-coil peptides

Introduction: Protein-like polymers comprised of covalently linked supramolecular peptidic structures represent the next-generation advanced materials that overcome the limitations of traditional synthetic polymers. Self-assembled coiled-coil peptides have been polymerized through thia-Michael addition in an end-to-end fashion to produce rigid polymers with persistence lengths up to 40 µm. We are interested in developing new hydrogel materials with tuneable composition and dynamic properties for biomedical applications. In this study, we identified conditions for the synthesis of high molecular weight polymeric coiled-coils (PCCs) via step-growth polymerization of coiled-coil peptides ("bundlemer") employing tetrazine ligation, the inverse electron demand Diels-Alder cycloaddition reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) derivatives. When the polymerization was conducted at high bundlemer concentrations, physically entangled hydrogels with robust mechanical properties and thermal responsiveness were obtained.

Materials and

Methods: Cysteine-tagged bundle-forming peptides (DE-N-Cys) were prepared by solid phase peptide synthesis (SPPS). Tetrazine or TCO was conjugated to the peptides via thia-Michael addition to afford the supramolecular building blocks (DE-Tz)4 and (DE-TCO)4 (Fig. 1) after dissolution in DI water. Step growth polymerization was carried out in aqueous media under strict stoichiometry at peptide concentrations of 0.1 to 10 mM to yield polymeric coiled-coil peptides. PCC-based hydrogels were produced when step growth polymerization was performed at peptide concentrations greater than 5 mM. The polymerization kinetics was monitored by measuring tetrazine absorbance at 524 nm by ultraviolet-visible spectroscopy (UV-vis). Dilute solution viscometry and diffusion-ordered spectroscopy (DOSY) were applied to assess the PCC diffusivity and molecular weight. Circular dichroism spectroscopy (CD) was used to characterize the thermal stability of the coiled-coils. Transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) were employed to characterize the PCC structure and morphology. A coarse-grain (CG) model was utilized to microscopically investigate the PCC conformation and Kuhn length. Finally, oscillation rheology was exploited to characterize the rheological properties of the PCC hydrogels.

Results, Conclusions, and Discussions: The Tz/TCO reaction is uniquely suitable for polymerization of self-assembled coiled-coil peptides due to its fast reaction kinetics (with a second order rate constant 103-106 M-1s-1), high selectivity at low concentration, biocompatibility, and exceptional yield. We have applied this chemistry to the synthesis of hyaluronic acid (HA)-based hydrogels3 with spatiotemporal gradients and poly (ethylene glycol) (PEG)-based microfibers with cell-adhesive properties.4 In this investigation, Tz and TCO groups were conjugated to the DE peptide via flexible PEG linkers. Long and semiflexible rods were fabricated through tetrazine ligation-mediated step-growth polymerization. Based on dilute solution viscometry and DOSY experiments, the apparent molecular weight of PCCs was estimated to be 100 – 3,000 kDa, depending on the bundlemer concentration during polymerization. Experimental observations and computational modeling collectively indicate the formation of polymeric chains with a Kuhn length of 6-7 nm. Polymerization at high bundlemer concentrations (> 5 mM) led to the establishment of physical gels through interchain entanglements of the semi-flexible protein-like polymers. The gelation time and stiffness of PCC gels were monomer concentration dependent. At a monomer concentration of 10 mM, the resultant PCC gels were mechanically robust, exhibiting an average elastic modulus of 400 Pa and a strain to failure of 300%. Analysis by oscillatory rheometry reveals the disruption of the network structure around the melting temperature of the coiled-coiled peptide. Upon cooling, partial recovery of the gel strength was observed.

Overall, the polymerization of novel coiled-coil peptides with biorthogonal tetrazine-TCO ligation gives rise to the construction of protein-like supramolecular-covalent hybrid polymers.

Acknowledgements (Optional): This research was primarily/partially supported by the National Science Foundation (NSF) through the University of Delaware Materials Research Science and Engineering Center DMR-2011824.