Preparation and characterization of biomimetic materials inspired by jumbo squid sucker ring teeth
Date of Issue2016-03-01
School of Materials Science and Engineering
Biological & Biomimetic Material Laboratory @ NTU
Dosidicus gigas (D. gigas, or Jumbo squid) sucker ring teeth (SRT) are newly discovered biological materials with intriguing combination of mechanical and physico-chemical properties. Notably, recent studies have shown that SRT are devoid of minerals, chitin and inter-chain covalent cross-links, and are entirely constituted of proteins called “suckerins” which form a polymer network reinforced by self-assembled nano-scale β-sheet structures. At the molecular (primary sequence) scale, suckerins display a block co-polymer sequence consisting of two tandem alternating modules. One is rich in alanine (Ala) and resembles the poly-Ala or poly(glycine (Gly)-Ala) domains of silk proteins and form β-sheets, and the other is dominated by Gly accompanied with high contents of tyrosine (Tyr) and leucine (Leu) residues. These characteristics make SRT a promising model for biomimetic materials research. In this project, therefore, we hypothesize that functional materials with β-sheet structures could be engineered with SRT proteins and these materials could be utilized in a broad range of biomedical and engineering applications that have been demonstrated for silks. Furthermore, the goal of the project is to further deepen the structure/property relationships of SRT, notably to link their thermo-mechanical response with the protein structure of suckerins, which is unknown prior to this work. We hypothesize such structure/property relationships would provide molecular scale biomimetic principles to develop a new range of protein- and peptide-based materials. Characterization of SRT by nanoindentation was conducted in different solvents that could disrupt their putative secondary structures. We found that β-sheets disruption in SRT was correlated to a concomitant decay of Young’s modulus (E), which provided the first direct evidence of hydrogen bonding localized in β-sheets as an essential contributor to SRT mechanics. This also explained the fundamental structure-property relationship of SRT. SRT and suckerins also displayed thermoplastic properties, where the crystalline β-sheets and amorphous domains exhibited different thermal stabilities and thermo-mechanical responses. These intriguing characteristics motivated us to fabricate materials with native SRT proteins, including melt-spun fibers and electro-spun nanofibers. The melt-spun fibers displayed polymer chain alignment and comparable mechanical strength to regenerated silk fibers. Electro-spun nanofibers were made by using an additive and a systematic optimization of electro-spinning parameters that led to smooth nanofibers. In order to sustainably and efficiently supply SRT proteins for materials engineering, a recombinant expression system for suckerin-19 was established. The production of two suckerin-19 variants was achieved and a facile chromatography-free purification method was developed, with promising protein yields. In order to demonstrate the hypothesis that recombinant SRT proteins were also able to produce materials, suckerin-19 was fabricated into tunable materials by a RuII mediated photo-cross-linking method. This method allowed the formation of hydrogels and films whose surface roughness, cross-link density and β-sheet content could be tailored. Importantly, the stiffness of these gels and films could be modulated over 7 orders of magnitude by varying the cross-linking conditions or by the addition of a common plasticizer. This tunability of E enables them to match the elasticity of a wide range of human tissues from soft liver to stiff bone, opening promising avenues in tissue engineering and restorative applications. Overall, this research demonstrates that suckerins’ characteristics, namely β-sheet self-assembly, modular sequence design, impressive mechanical properties and biocompatibility, provide a novel toolbox of protein-based materials to biomaterial scientists, with a broad spectrum of potential biomedical and engineering applications.