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Bioorthogonal synthesis of complex macromolecular assemblies and functional biomaterials
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The need for advanced materials with tunable properties and adaptive features has motivated researchers to exploit the utility of peptidic building blocks and motifs to create new materials with desired functions. Meanwhile, hydrogel biomaterials that recapitulate the spatial heterogeneity and temporal dynamics seen in the native extracellular matrix (ECM) have become indispensable for the successful engineering of functional tissues or tissue models. Current methods for biomaterial synthesis employ toxic reagents or non-physiological triggers, are of low efficiency, and interfere with native biology. Tetrazine ligation, an inverse electron-demand Diels–Alder (IEDDA) cycloaddition between s-tetrazines (Tz) and strained alkenes, features high yield, high selectivity, tunable rates, biocompatibility, and bioorthogonality, thus desirable for use in biomaterials synthesis. My dissertation aims to apply tetrazine ligation to synthesize functional biomaterials and complex macromolecular assemblies. ☐ To establish a synthetic matrix that can be modified in situ during 3D cell culture, I exploited the differential reactivity of norbornene (Nb) and trans-cyclooctene (TCO) towards tetrazine. While Nb engages with Tz with an apparent second-order rate constant (k2) of 100 M-1 s-1, TCO reacts with Tz ferociously (k2 ~ 105 M-1 s-1), exceeding molecular diffusion through hydrogel materials. Cell-laden hydrogel was initially fabricated by reacting tetrazine-modified hyaluronic acid with a Nb-tagged, protease-cleavable peptide crosslinker at a Tz/Nb ratio of 2/1. After a few days of culture, matrix adhesiveness or stiffness were altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining Tz groups in the network at the gel–liquid interface. Because the Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. The dynamically tunable hydrogel system was applied to investigate how in situ modulation of matrix adhesiveness induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. ☐ Separately, protein-like hybrid polymers consisting of covalently linked coiled-coil microdomains with regularly spaced ethylene glycol repeats via tetrazine ligation-mediated step-growth polymerization. Polymerization of Tz and TCO-functionalized peptides in aqueous media under strict stoichiometry at Tz or TCO concentrations of 0.1 to 4.5 mM leads to the establishment of exceptionally long, semiflexible polymer chains with a Kuhn length of 6–7 nm and an apparent molecular weight up to 3 MDa. Bioorthogonal polymerization at bundlemer concentrations above 5 mM gives rise to physical gels through interchain entanglements. Hydrogels prepared at 10 mM exhibit an average elastic modulus of 400 Pa and a strain to failure of 300%. Copolymerization of coiled-coil peptides with distinct composition and thermal stability results in hydrogels that are thermally tunable. A solid-to-fluid transition occurs when one of the coiled-coil repeats melts. Upon cooling, solid-like properties are partially recovered through the intermolecular association of the helical peptides. ☐ Overall, this work demonstrates the utility of tetrazine ligation for the development of innovative materials with unprecedented control over polymer composition and molecular weight, as well as the dynamic tunability of ECM-mimetic hydrogels.
Description
"At the request of the author or degree granting institution, this graduate work is not available to view or purchase until August 31 2026."--ProQuest abstract/details page.
Keywords
Biomaterials, Hydrogel, Peptides, Supramolecular assembly, Extracellular matrix