The Fibrillization And Physical Gelation Of Bombyx Mori Silk Fibroin

Through millions of years of adaptation and evolution, spiders and silkworms made'perfect'fibers rich in?-sheet structures, which are well known as animal silks. After half a century of intensive study, the full hierarchical structures of animal silks, from the primary structure (i.e., the amino acid sequence) to the condensed-state, have been partially uncovered. Based on such knowledge about the hierarchical structures, researchers have made various silk-protein based materials. On the other side, amyloid fibrils primarily associated with diseases such as Alzheimer's, are the type of?-sheet-rich protein aggregates as well. As many proteins which are not implicated in disease can be induced to form amyloid-like fibrils with?-sheet-rich state, the candidates for the fabrication of structural protein based materials are greatly enlarged. However, how to turn the amyloid-fibril-like materials with various nanostructures into the applicable macroscopic materials still remains a lot of challenges.The present thesis was focused on the aspect of fibrillization and physical gelation of Bombyx mori silk fibroin (SF). An extensive exploration of the assembly of?-sheet at higher hierarchical levels was fulfilled. Also, a preliminary discussion on the strategy employed by Bombyx mori silkworm to prevent the gelation and solidification of silk fibroin in the silk gland before spinning was addressed. Thus, our work was expected to provide an inspiration to the structural control in the development of?-sheet-rich protein materials In the first part of this work, it was showed that Bombyx mori silk fibroin could be selectively induced to fold into two distinct fibrils with alternative?-sheet structures, i.e., cross-?structure (where the?-strands arrange perpendicular to the fibril axis), as incubated in ethanol-water quiescently or parallel-?structure (where the?-strands arrange parallel to the fibril long axis), as incubated in water under shear. Further, it was found that the 'continuous'?-sheet in the cross-?fibril was composed of at least 15?-strands, whereas in the parallel-?fibril, most individual?-sheet, intervened by segments with non-?-sheet structure or less-ordered?-sheet structure, was composed of 4 to 10?-strands. We also proposed a tentative structural model for the cross-?fibril based on the amino acid sequence of silk fibroin. The method presented, i.e., using mechanical force to modify fibril structure, might afford a new approach to design of novel self-assembling?-sheet protein materials.It was further studied the hierarchical structure and formation kinetics of SF alcogel developed from cross-?fibrils. The results showed:(1) The structure of SF alcogel could be understood on two levels, i.e., SF fibrillar aggregates (clusters) on the nanoscale and floc-linked network on the microscale. (2) Fibrillar clusters could be recognized as a ramified fractal with dimensions(Df) varying from 2.4-2.8, depending on the SF concentration. The floc-linked network, with Df of 2.2, was formed through the aggregation of the fibrillar clusters. (3) Regarding the fibrillization mechanism, our results suggested that the SF underwent a nucleation-growth process. The increase of ethanol concentration could speed up the nucleation process. Branching and twisting of growing fibrils gave rise to the formation of fibrillar clusters. (4) The concentration of SF played a crucial role in gelation kinetics, elastic modulus as well as strain hardening property under large amplitude shear oscillation of the gel.A novel blend hydrogel comprising SF and hydroxypropylcellulose (HPC, a thermosensitive polysaccharide) was developed therefore. The blends gelled at 37?, and the hydrogel showed thixotropic property, which is injectable. According to the analysis of dynamic rheological measurements, confocal laser scanning microscopic images, Raman spectra and quantitative 13C NMR spectra etc., a possible gelation mechanism was assumed as follows:(1) When the SF-HPC blend solution was heated from room temperature to 37?, the hydrophobic interaction between HPC chains enhanced, triggering the phase separation of the blend. The phase separation induced the emergence of SF-rich phase, and thus initiated the conformational transition of SF from random coil to?-sheet. (2) As phase separation proceeded, the phase-separated microstructure coarsened. Meanwhile, the P-sheet content of SF increased, and ultimately, a percolated network formed. (3) The?-sheet content of SF continued increasing after gel point, leading to the ripening of the gel network and immobilizing the dispersed phase and the molecular chains therein. The composition of the blend was found to have important effect on the kinetics of both phase separation and gelation, as well as the final morphology and mechanical property of the hydrogel. Also, the hydrogels with different composition showed distinct thixotropic properties. Based on the flow behavior of the hydrogel under large amplitude oscillatory shear (LAOS), the underlying mechanism of shear-thinning and immediate recovery was discussed. The proposed scenario of the LAOS behavior went as follows:1) the elastic deformation between?-sheet-rich domains;2) the destruction of the weak crosslinks between?-sheet-rich domains; 3) the slip of?-sheet-rich domains; 4) the reconstruction of the weak crosslinks. Moreover, the hydrogel was demonstrated to encapsulate cells (e.g. fibroblasts L929), and to protect the encapsulated cells against high shear force during injection. Thus, the injectable SF-HPC blend hydrogel as a minimally invasive formulation may find applications in numerous biomedical fields, such as cell therapy and drug delivery.The investigation of rheological properties of'sol-like'natural spinning dope of Bombyx mori silkworm was also involved in this work. It was found:(1) The lyotropic spinning dope showed flow-tumbling behavior, resulting in the unusual rheological properties, such as a negative first normal stress difference (N1), and transient oscillations of viscosity (or shear stress) and N1 at moderate shear rates. (2) The reactive parameter|?|, which is the character of the flowing type of nematics, was determined to be.0.996, suggesting a largely anisotropic shape of silk fibroin in the liquid crystalline phase. (3) A supramolecular mesogen was assumed to give rise to the formation of liquid crystal, and three states of fibroin molecules in the spinning dope upon shear was suggested as:1) a transiently associated network, the modulus of which lied in the range of 1.2 X 104-2.0×104Pa and the relaxation time of which lied in the range of 0.5-3s, existed in the absence of shear; 2) the transient network partially unassociated upon shear, leading to a significant decrease in the modulus and 3) further increased shear destructed the transient network, and resulted in the tumbling of the supramolecular nematics. Finally, it was speculated that the tumbling behavior of SF nematics at moderate shear rates might play a role in stabilizing natural spinning dope and preventing SF from conformational transition (to a?-sheet-rich state) and solidification within silk gland.