PROGRAM | Materials Science & Engineering

By: Sai Shreedhar Patkar Chair: Kristi Kiick

ABSTRACT

Modulation of inter- and intramolecular interactions between bioinspired designer molecules can be utilized for developing functional structures that mimic the complex hierarchical organization of multicomponent assemblies observed in nature. Furthermore, such multistimuli-responsive molecules offer orthogonal tunability for generating versatile multifunctional platforms via independent biochemical and biophysical cues. This dissertation harnessed the remarkable mechanical and physicochemical properties of intrinsically disordered resilin-like polypeptides (RLPs) for generating nanostructured and microstructured biomaterials via their stimuli-responsive phase separation.

The native extracellular matrix (ECM) is characterized by structural and compositional heterogeneity across various length scales. Current state-of-the-art in soft-matter patterning techniques to produce sophisticated ECM mimics are limited due to their reliance on expensive equipment and multiple time- and energy-intensive steps. In the first aim of this work, opportunities to produce bi-component elastomeric matrices by exploiting the liquid-liquid phase separation (LLPS) of RLPs are reported. Temperature-triggered LLPS of RLPs can be precisely controlled by altering their amino acid composition. Hence, the photocrosslinking of distinct morphologies of multicomponent RLP solutions during LLPS presents exciting opportunities for developing a simple biofabrication method. This research enabled fine-tuning of pore diameters and shear storage moduli of microstructured hydrogels by manipulating sequence-encoded differences in phase separation of multicomponent RLP formulations.  The developed microstructured hydrogels can be utilized as cell-instructive materials to support regenerative medicine applications.

While RLPs can be stimuli-responsive, the disorder encoded in their secondary structure has limited their usage for creating structurally defined nanoassemblies. Hence, there exist no studies with the use of short RLP-based sequences for generating stimuli-responsive materials which are ordered on the nanoscale. In contrast, computationally designed coiled-coil peptides or ‘bundlemers’ have demonstrated versatility for producing programmable 1D, 2D, and 3D nanostructures via covalent assembly of peptide bundles with complementary functional groups. To afford them physical responsiveness, for the second aim of this work, two different bundlemer- forming peptides were functionalized with thermoresponsive RLPs of various lengths to induce hierarchical nanostructure assembly via the temperature-triggered phase separation of the RLP motifs. Genetic fusion to the coiled-coil bundles not only displayed the short RLPs in an ordered fashion, but also substantially increased their transition temperature, below which the ordered display of collapsed RLPs promoted 1D-like assembly into nanofibers.

Extending the research findings from the first two aims, bundlemer-forming peptides were fused to the N- and C- termini of compositionally distinct yet similar RLPs. The resulting genetically fused conjugates exhibited improved thermal stability and retained sequence-encoded control over thermoresponsiveness. The influence exerted by the ordered helical domains on the RLPs and on each other, could be further altered by manipulating the phase separation of RLPs in response to additional environmental stimuli such as pH, salt identity, and ionic strength. Transmission electron microscopy indicated that assembly at high temperature (80°C) mediated the formation of ribbon-like structures.

These investigations helped to establish the design parameters for sequence-encoded control over inter- and intramolecular interactions of ordered and disordered building blocks to create hybrid and responsive materials with defined nano- and microstructures.

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