Abstract
Silk fibroin produced by the domesticated silkworm, Bombyx mori, has been studied widely as a substrate for tissue engineering applications because of its mechanical robustness and biocompatibility. However, it is often difficult to precisely tune silk fibroin’s biological properties due to the lack of easy, reliable, and versatile methodologies for decorating it with functional molecules such as those of drugs, polymers, peptides, and enzymes necessary for specific applications. In this study we applied an azido-functionalized silk fibroin, AzidoSilk, produced by a state-of-the-art biotechnology, genetic code expansion, to produce silk fibroin decorated with cell-repellent polyethylene glycol (PEG) chains for controlling the cell adhesion property of silk fibroin film. Azido groups can act as selective handles for chemical reactions such as a strain-promoted azido-alkyne cycloaddition (SPAAC), known as a click chemistry reaction. We found that azido groups in AzidoSilk film were selectively decorated with PEG chains using SPAAC. The PEG-decorated film demonstrated decreased cell adhesion depending on the lengths of the PEG chains. Azido groups in AzidoSilk can be decomposed by UV irradiation. By partially decomposing azido groups in AzidoSilk film in a spatially controlled manner using photomasks, cells could be spatially arranged on the film. These results indicated that SPAAC could be an easy, reliable, and versatile methodology to produce silk fibroin substrates having adequate biological properties.
Highlights
Silk fibroin is a huge protein polymer produced by the domesticated silkworm Bombyx mori.It is regarded as a heterodimer of fibroin heavy chain (FibH; ~390 kDa) and fibroin light chain (FibL; ~26 kDa) [1,2]
We demonstrated the power of genetic code expansion methodology to prepare silk fibroin for decoration by strain-promoted azido-alkyne cycloaddition (SPAAC) with functional molecules in a biocompatible manner
AzidoSilk, where the azido-bearing synthetic amino acid AzPhe is incorporated in the primary structure of silk fibroin, for the decoration with polyethylene glycol (PEG) by SPAAC
Summary
Silk fibroin is a huge protein polymer produced by the domesticated silkworm Bombyx mori. A research group at NARO first applied the genetic code expansion methodology to B. mori in order to create silk fiber with novel functions [15] They succeeded in efficiently incorporating an azido-bearing synthetic amino acid, 4-azido-l-phenylalanine (AzPhe), into silk fibroin in collaboration with a research group at RIKEN [16]. AzidoSilk production was scaled up by a simple hybridization of a transgenic line that has the ability to incorporate AzPhe in silk fibroin with a high-silk-producing strain [21] This achievement, combined with various functionalization approaches by click chemistry reactions, would facilitate industrial applications of silk fibroin materials with specific functions. By partial UV irradiation through photomasks before click reactions with PEG, the spatial arrangement of cells on AzidoSilk film was achieved These achievements by the power of genetic code expansion would lead to the production of self-standing, biocompatible, and spatially arrangeable silk-based cell substrates for tissue engineering applications
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