ATR-FTIR analysis of SF films
Infrared absorption spectra of water annealed and GSH modified SF film show characteristic absorption bands assigned to the peptide bonds (–CONH–) that originate bands known as amide I, amide II, and amide III (Fig. 2a). It can be observed from FTIR spectra that amide I, amide II, and amide III of both water annealed and GSH grafted SF film were at 1645 cm−1, 1522 cm−1, and 1235 cm−1, respectively. Therefore, it can be concluded that GSH modification had no significant effect on the secondary structure of the silk fibroin film.
In addition, amide I is useful for the analysis of the secondary structure of the proteins and is mainly related with the C=O stretching, and it occurs in the range of 1700–1600 cm−1. The water annealed SF film spectra shows a peak at 1645 cm−1, suggesting the presence of the helical conformation [22], with a shoulder at about 1629 cm−1, indicating a level of β-sheet conformation [23], for amide I. FTIR spectra of GSH-modified SF film showed a peak at 1645 cm−1 with a strong shoulder at about 1629 cm−1 for amide I as well, which reflects a similar molecular conformation as that of water annealed SF film (Fig. 2b).
FTIR spectroscopy also provides evidence that GSH was grafted onto SF film. The absorption band at 3276 cm−1 represents for N–H stretching vibration of amide [24], and the intensity of this band after carbodiimide coupling is apparently increased indicating the increased density of the amide bond (Fig. 2c), which is from the reaction between primary amine of reduced glutathione (GSH) and activated carboxylic groups in silk fibroin, or vice versa.
XPS analysis of SF films
Figure 3 showed XPS analysis results of the surface S 2p high resolution spectra of a water annealed (Fig. 3a) and a GSH-modified (Fig. 3b) SF films. Compared with water annealed sample, a new peak for the elemental 2p sulfur was detected on the surface of GSH-modified one. It can thus be concluded that sulfur component is present on the surface of SF films, therefore confirmed the successful installation of GSH on the surface of SF films. Deconvolution of XPS S 2p spectra was performed to estimate chemical state of thiol groups. A typical curve fitting of the S 2p peak region is shown in Fig. 3b. The doublet at 163.1 eV is consistent with the formation of disulfide bonds [25]. Therefore, a disulfide bond reducing agent is required in order to access the free thiol groups on SF film.
In order to confirm the reactivity of thiol groups on GSH-modified silk fibroin film, the sample was kept in 10 mM TCEP·HCl solution for 20 min at room temperature followed by rinsing with ultrapure water thoroughly. The sample was then incubated in 10 mM AgNO3 solution for 10 min, followed by sonicating in ultrapure water for 5 min to remove bound AgNO3 and rinsed thoroughly with ultrapure water. Figure 4a shows the XPS survey spectra of GSH-modified SF film after soaking in AgNO3 solution, where the typical peaks assigned to C 1s, N 1s and O 1s can be observed at 285 eV, 400 eV and 532 eV clearly [26]. The results of the XPS spectrum in Fig. 4a also confirmed the existence of Ag (368 eV) on the sample surface, suggesting the existence of interactions between SF and Ag. Then, a high-resolution XPS spectrum of Ag 3d was recorded. The core level binding energy of Ag 3d was observed as 374.2 and 368.2 eV which is attributed to the Cys-capped Ag [27]. This means that Ag was linked to the Cys of GSH grafted onto the silk fibroin film surface, indicating the presence of metal–thiolate bonding and illustrating the reactivity of the HS-SF film. In addition, the potential use of Ag/SF film composite as antibacterial material was assessed by observing their antibacterial activity against E. coli. The antibacterial properties of silver-loaded SF films were evaluated by disc diffusion assay. It was found that silver-loaded SF films can effectively inhibit the growth of E. coli (Additional file 1: Figure S1).
Hydrophobicity
The contact angles measured on the water annealed silk fibroin films and the GSH-modified silk fibroin films were presented in Fig. 5. Smaller contact angle usually indicates that the material surface is more hydrophilic, enhancing the cell adhesion and proliferation. The contact angle for the water annealed silk fibroin film was 57.9 ± 3.0°. A significant difference (p < 0.05) was detected after the water annealed silk fibroin film was treated with carbodiimide coupling reaction, which had the contact angle of 46.5 ± 4.4°. Compared to the water-annealed silk fibroin films, the decrease of contact angle may be attributed to the covalent coupling of hydrophilic peptides.
Morphology
AFM was used to exam surface morphologies of samples, and root-mean-square (RMS) values were calculated to determine surface roughness of samples. For water annealed SF film, granular features with a lateral dimension of 49.2 ± 3.6 nm totally covered its surface (Fig. 6a). Those aggregated granules were densely grouped together. GSH-modified SF film was very similar to water annealed SF film but the granules were much larger than those formed on the surface of water annealed SF film with a lateral dimension of 59.2 ± 4.7 nm (Fig. 6b). The surface roughness was increased from 1.6 ± 0.1 to 2.4 ± 0.3 nm (n = 3). This result suggested that the peptide covalent coupling to the surface of SF films increases their surface roughness.
Structure analysis
To detect if changes in the crystalline structure were induced by carbodiimide coupling, X-ray diffractometer (XRD) profiles of the silk fibroin film before and after the carbodiimide coupling reaction were examined (Fig. 7). The principal diffraction peaks of the Silk I crystal structure (random coil content) are 12.2°, 19.7°, 24.7° and 28.2°; the diffraction peaks of Silk II crystal structure (β-sheet content) are 9.1°, 18.9° and 20.7° [28,29,30].
Both X-ray diffraction curves of water annealed and GSH-modified SF films show diffraction peaks at 2θ = 12.2° and 20.7°. Above data indicate some silk I structure and silk II structure are in these films. Silk I conformation can be explained by slow crystallization for silk fibroin from liquid during film casting at ambient conditions [31]. While water annealing process can gradually change SF film’s crystal structure from silk I to silk II [30]. No obvious change in the crystalline structure was observed from the X-ray diffraction of silk fibroin film after carbodiimide coupling reaction.
Biocompatibility
The MTT assay is a common method for evaluating biomaterial toxicity based on the mitochondrial activity, which influences metabolic activity and cell viability. To determine the toxicity profile of the GSH-modified silk fibroin film, we conducted the standard MTT cytotoxicity assay with HEK293 cells [32, 33].
As Fig. 8 demonstrates, the cells were treated with leaching liquors from water annealed SF film and GSH-modified SF film, as well as phenol solution with various concentrations respectively, which were coded as C0, 1/2C0, and 1/4C0 (Fig. 8). Comparing the leaching liquor from water annealed SF film, the leaching liquor from GSH-modified SF film decreased the HEK293 cell viability slightly. This difference perhaps to be relative with the presence of EDC trace residue within the sample, and the previous study demonstrated that EDC had negative effects on the cell viability [34]. Apart from that, both water annealed and GSH-modified SF films exhibited high biocompatibility as their cell viabilities were higher than 100% relative to control. In addition, the HEK293 cell viability of both water annealed and GSH-modified SF films was slightly decreased after their leaching liquors were diluted. Because it is a dose dependent continuous decrease, we could conclude that silk fibroin based material can enhance cell proliferation.
HEK293 cell adhesion on surfaces of water annealed and GSH modified SF films were observed to better understand how GSH modification can play a role in cell adhesion and spreading (Fig. 9).
At day 1, optical microscope image indicated that cell adhesion was randomly distributed on both water annealed and GSH modified SF films. In addition, certain number of cells appeared in an elongated shape to GSH modified SF film, but with a widening morphology, or very flat, on water annealed SF film. This might because a smoother surface (water annealed SF film) can help in the very first states of cell adhesion.
Three days after seeding, the cells that adhered to water annealed and GSH modified silk fibroin film were comparable both in terms of number and morphology. In addition, at this time, individual cells were in direct contact with other cells forming a monolayer. These data indicate that GSH-modified SF films are biocompatible and have no adverse influence on the growth of the HEK293 cells.