Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/77361
Title: Study of recombinant spider eggcase silk fibers and spheres
Authors: Chen, Jianming
Advisors: Hu, Jinlian (ITC)
Yang, Mo (BME)
Keywords: Silk
Fibers
Issue Date: 2018
Publisher: The Hong Kong Polytechnic University
Abstract: Spider silks, assembled under different combination of proteins, have been attractive biopolymers with extraordinary mechanical properties and biomimetic potentials. Thereof, spider eggcase silk, as a task-specific fiber, draws increasing attention due to its unique properties. Tubuliform spidroin 1, from a black widow spider-Lactrodectus Mactans, was genetically engineered by using the single repeat unit and abbreviated as eTuSp1. In this work, highlights would be shed on the study of: a, silk assembly mechanism of eTuSp1 and its micellar structure; b, facile fabrication of silk spheres in a lubricant-based system; c, engineered silk spheres as potential platform for drug delivery applications. By the analysis of amino acid sequence, an amphiphilic silk protein with distinct hydrophobic and hydrophilic domain was detected by the hydropathy plot. To support the theoretical estimation for micelle-like structure, the synchrotron radiation SAXS was combined with DLS and TEM to reveal the formation of micelles and soluble aggregates for the eTuSp1 in HFIP. The assembly mechanism of silk spheres and fibers was investigated by a microfluidic device, which was capable of mimicing the physical and chemical conditions as the natural spinning process. To replace the function of ions exchange and acidification, the desolvation of the highly conserved eTuSp1 was simplified by the non-solvent induced phase separation in the absence of N-/C-terminus. Solely by addition of isopropanol, the eTuSp1 was assembled as spheres, predominantly obtaining a helical conformation. In good agreement with previous studies, spherical aggregates were considered as intermediates and could be induced into ß-sheet-rich silk fibers by the shear and elongation. The underlying mechanism for the assembly of silk spheres and fibers were demonstrated by the micelle theory. To investigate mechanical properties, individual spheres were subjected to the AFM indentation and the corresponding compressive modulus was determined by using Hertz model. In the tensile test, the modulus of silk fibers could be flexibly regulated in accordance to different post-spin drawing ratios. Inspired by the natural spinning within spiders, various lubricants were explored to show its effect on the formation of silk proteins in various morphologies, like spheres, capsules and films. By using the suitable surface tension and viscosity, silk spheres were assembled as rapidly as 10 s under a lubricant-based system. The fabrication of silk spheres follows the theory of slovent-induced phase separation. The obtained particles with spherical shape were mainly attributed to the interfacial emulsification and the micelle structure. With regard to the increase of protein concentration, the average dimension and size distribution of silk spheres were both increased. Evidenced by FTIR, the freshly-made spheres adopted as the same helical conformation as the eTuSp1 in HFIP. And upon exposure to ethanol treatment, secondary structure of silk spheres could be effectively changed to ß-sheet-rich conformation. The thermal properties of eTuSp1 were also discussed by the DSC and TGA. Silk spheres are frequently investigated as carriers in reponse to certain stimulus for drug delivery application. In the presence of proteinase K, ethanol-mediated silk spheres exhibited distinct degradation behaviors by the aid of CLSM, revealing a tunable enzyme-triggered drug delivery. In addition, the pH-sensitive silk spheres were also exploited. To optimize the electrostatic interaction for lysosomal drug delivery, the spider-eggcase-silk protein was genetically engineered using 5×His Tag with a tailor-made isoelectric point of 4.8. Followed by the post-treatment, silk spheres obtained an improved mechanical integrity, which was specifically determined with the compressive modulus of 9.6 MPa by AFM indentation. Significantly, the accumulative release of doxorubicin by 96 h was approximately 4.5-fold higher at pH 4.5 than that at pH 7.4. To further demonstrate this release behavior on Hela cells, within 24 h, doxorubicin-loaded silk spheres were first observed to accumulate in lysosomes and then efficiently released the payload in response to the trigger of lysosomal pH.
Description: xxix, 170 pages : color illustrations
PolyU Library Call No.: [THS] LG51 .H577P ITC 2018 Chen
URI: http://hdl.handle.net/10397/77361
Rights: All rights reserved.
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