Due to their extraordinary mechanical and biochemical properties, spider silks represent fascinating protein-based materials which are promising candidates for versatile biomedical and technical applications. In vivo, fibers are formed from silk proteins. In vitro, however, recombinantly produced spider silk proteins can be assembled into different morphologies, including particles, capsules, non-wovens or films. Here, films made of the recombinant spider silk protein eADF4(C16), which is based on the sequence of the repetitive core domain of the dragline silk protein ADF4 of the European garden spider Araneus diadematus, are characterized. The influence of different processing parameters including the initial solvent of the protein (hexafluoroisopropanol, formic acid and aqueous solution), the used post-treatment method (methanol and ethanol as well as potassium phosphate) and relative humidity on distinct film properties are systematically studied. While all tested parameters influenced the secondary structure of the proteins within the films, they had no remarkable effect on the films’ thermal stability, as determined by DSC and TGA measurements. In contrast, chemical stability and mechanical characteristics revealed a clear dependence on the initial solvent and post-treatment, and could be correlated with the secondary structure content in the film. Tensile testing, dynamic mechanical analysis, as well as combined polarized IR spectroscopy and mechanical analysis indicated an influence of the initial solvent not only on the presence but also on the arrangement of secondary structure elements, with the effect being sustained even after methanol treatment. Analysis of surface characteristics of the different films revealed an influence of the tested post-treatment procedures on the films topography and hydrophobicity. In contrast, the initial solvent only showed minor effects, except for the aqueous system. For many applications specific functionalization with external effector molecules is necessary or beneficial. Therefore, post-translational chemical coupling was chosen. In order to provide a reaction site for specific and controlled functionalization, cysteine containing variants of eADF4(C16) were produced. Three different cysteine variants, containing a single cysteine each, were characterized in terms of secondary structure, assembly and chemical reactivity in solution. Their structural characteristics were comparable to the original cysteine-free protein both in solution and in assembled films. Further, the proteins in solution as well as in cast films could be easily modified. Chemical coupling with molecules like nanogold, fluorescein, biotin or beta-galactosidase demonstrates the possible site-selective functionalization of silk films. Additionally, a cyclic RGD-peptide was added to the films in order to enable integrin-mediated cell adhesion in subsequent biocompatibility tests. With respect to potential future applications of the spider silk films, different technically important hydrophobic (polystyrene and Teflon) and hydrophilic (glass) substrates were coated with eADF4(C16). As critical parameters, surface properties like topography and wettability were analyzed. It was shown that especially the wettability of thin silk films depended on the chosen substrate, with intrinsically hydrophobic substrates revealing a hydrophilic film surface and vice versa. Additionally, changes in the secondary structure of the proteins were observed in these different films. Therefrom, a model concerning the arrangement of the proteins/protein motifs within the film was derived, according to basic principles of block-copolymer chemistry. Due to the biocompatibility of natural silks, silk materials are interesting candidates for use in medical applications. Therefore, the biocompatibility of eADF4(C16) films was investigated. Pre-osteoblasts (MC3T3-E1) could be cultured on the silk films for several days revealing no adverse effects. The initial solvent used for film preparation and the used post-treatment method influenced cell behavior, which can probably be attributed to differences in surface chemistry and topography of the respective films. However, adhesion of the cells on non-modified films seemed to be weak, which could be improved by coupling of the cyclic RGD-peptide c(RGDfK) to the films. Overall, this work provides information about how distinct properties of recombinant spider silk films may be influenced and controlled by varying processing parameters. The results reveal insights into the structure-function-relationship of silk proteins in such films, which provides basic knowledge for future investigations directed to specific applications thereof. The possibility of versatile functionalizations in combination with their general biocompatibility indicates a high potential for several applications of films made of recombinant spider silk proteins in technical as well as biomedical fields.
|Commitee:||Schmid, Franz X., Fery, Andreas, Senker, Jürgen|
|School:||Universitaet Bayreuth (Germany)|
|Source:||DAI-C 81/4(E), Dissertation Abstracts International|
|Keywords:||Recombinant spider silk proteins|
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