Effects of Ultraviolet Light Irradiation on Silk Fibroin Films Prepared under Different Conditions

Abstract

Silk fibroin (SF)-based materials are exposed to both natural and artificial ultraviolet (UV) light during preparation or administration. However, the effects of UV irradiation on SF films prepared under different conditions have not yet been described in detail. In this study, four SF films with different molecular weight (MW) distribution were fabricated using SF solutions, which were prepared by dissolving degummed SF for 0.5-24 h. We observed UV (365 nm) irradiation on SF films induced the increase of yellowness and absorbance at 310 nm of SF films, indicating the formation of new photo-products and di-tyrosine bonds by photo-oxidation. Due to di-tyrosine cross-links between SF chains, UV-irradiated SF films were not fully dissociated in urea solution. In addition to formation of new products, UV reduced the crystallinity of SF films by breaking hydrogen bonds of β-sheet conformation. Unlike the UV-induced decomposition of physical interactions, UV did not affect the covalent bonds (i.e., peptide bonds). Through these experiments, we could expect that SF with higher MW was more susceptible and SF with lower MW was more resistant to UV-induced photo-oxidation and photo-degradation. These results provide useful information about UV-induced aging of SF-based materials under natural sunlight and UV irradiating conditions.

Keywords: photo-degradation; photo-oxidation; silk fibroin film; ultraviolet aging.

Figure 1

SDS-PAGE results of four silk fibroin (SF) solutions. (A) Silver-stained gel image showing the difference of SFs in molecular weight distribution. The lane on the right side indicates the molecular weight marker. The other four lanes represent SF with increasing dissolution time, from left to right. (B) The gray value profiles of protein bands on the SDS-PAGE gel. The set of intensity values is taken from each lane, from the top to the bottom, and plotted in the chart.

Figure 2

Yellowness and total color differences (ΔE) of UV-irradiated SF films (1–4 h) prepared with four SF solutions that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n > 10; mean ± SEM).

Figure 3

Absorbance spectra of UV-irradiated SF films (1–4 h) prepared with four SF solutions that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h).

Figure 4

Morphologies of the remaining four SF films (A) and fluorescence intensity of the released SF molecules from SF films (B) after immersion of films in 6 M urea solution for 24 h (ex. 360/40; em. 460/40) (n = 3; mean ± SD; 1-fold: 0 h UV irradiation).

Figure 5

FT-IR spectra of SF films nonirradiated and irradiated with UV light for 4 h; (A) amide I region (1680–1600 cm⁻¹), (B) amide III region (1280–1200 cm⁻¹).

Figure 6

Amide I and III crystallinity index calculated from FT-IR spectra of UV-irradiated SF films prepared with four SF solutions that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n > 6; mean ± SEM).

Figure 7

Absorbance at 340 nm obtained by 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay with UV-irradiated SF films prepared with four SF solutions. The SFs were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n = 3; mean ± SD).