(15e) A Kinetic Path to Hierarchically Organized Soft Materials from Self-Assembling Discotics

Introduction: Proteins and other structural building blocks in nature traverse complex free energy landscapes to create hierarchically organized materials. For instance, collagen is a ubiquitous biopolymer which underlies structure and function in tissues. Many variants of collagen begin with triple-helical folding and further self-assemble into fibrils and higher order bundles. These end-state structures impart biological functions critical to the extracellular matrix (ECM) that surrounds cells, offering binding and receptor sites for various endogenous proteins and other biomolecules. Of note, the self-association pattern of the collagen triple-helix has been shown to be dependent on tissue environment and collagen protein type, generating a diversity of functional forms ranging from anchoring fibrils to vitreous gelators, guided by supramolecular cues directing the assembly pathway. One particular outcome of interest is the large laterally bundled network components that comprise the ECM, which facilitate cell binding, differentiation, and growth. We developed a model discostic amphiphile gelator based on the benzene tricarboxamide (BTA) supramolecular motif, which undergoes pathway-dependent structural maturation into triple-helices similar to that of collagen and collagen-like proteins, under varying rate of pH control as the guiding stimulus.

Methods: Isopeptides were synthesized with solid phase methods and conjugated to trimesoyl trichloride to yield isopeptide-bearing benzene tricarboxamide molecules, of which the three-arm species was isolated using HPLC. Hydrogels were then prepared via acidification of the samples mediated by either hydrochloric acid (fast) or glucono-δ-lactone (slow), and divalent calcium ions were contributed by either calcium chloride (fast dissociation) or calcium carbonate (slow dissociation). Gelation kinetics and viscoelastic properties of these hydrogels were investigated by oscillating rheometry. Circular dichroism spectroscopy was used to track the emergence of distinctive chiral signatures arising in the materials over time due to intrinsic chiral features ascribed to self-assembled individual stacks and triple helical fibrils. Transmission and scanning electron microscopy were employed to study the morphology of the self-assembled structures comprising the hydrogel. Polarized optical microscopy was used to study potential birefringent character imparted by the liquid crystalline nature of the hydrogels.

Results: Through slow pH change, complex hierarchical assemblies result, characterized by mesoscale elongated rod-like bundles embedded in a percolated mesh of narrow triple-helical fibers. In contrast, in the case of rapid pH change, the formation of such mesoscale superstructures is very minimal or non-existent. Furthermore, the presence of divalent cations in the system proved impactful, with markedly different outcomes arising in the cases of quickly vs slowly dissociating salts vs no divalent cation source, suggesting another dimension of dynamicity exhibited in this system. These cations were interpreted to play a critical role in mediating electrostatic bridging / charge screening that enables lateral bundling as well as one-dimensional growth, to different extents. All rheological hydrogels arising from these catalyzed self-assembly processes bear shear-thinning, self-healing features that would facilitate ease of passage through a syringe needle and subsequent restoration of gel state upon cessation of shear. The storage modulus of these hydrogels was furthermore dependent on the assembly pathway, with emergent hierarchical organization and lateral bundling conferring increased mechanical stiffness.

Conclusions: The broad range of heterogeneity in materials prepared from a single self-assembling gelator implies a measure of thermodynamic pathway control imparted by the gelation stimulus selection, and also suggests foreseeable merit in the pursuit of a collagen-mimetic biomaterial constructed from BTAs as a base unit. Their shear-thinning self-healing nature enables uses as an injectable ECM-mimicking material that could have application in regenerative engineering or cell-based therapy. Finally, modification of BTA substituents enables a catalogue of material building blocks analogous to the collagen family of proteins, where selection of one or more BTA type could give rise to a distinct material outcome.