The Fabrication of Cassava Silk Fibroin-Based Composite Film with Graphene Oxide and Chitosan Quaternary Ammonium Salt as a Biodegradable Membrane Material

2.1 Materials

Raw cassava silk fibers were purchased from the Dandong border economic zone cooperation Baoli Industrial Co., Ltd. (Dandong, China). GO was purchased from Xfnano Materials Tech Co., Ltd. (Nanjing, China). Protease-XIV and glycerol were purchased from Sigma-Aldrich trading Co., Ltd. (Shanghai, China). HACC was purchased from Nantong Lushen Bioengineering Co., Ltd. (Nantong, China).

2.2 The preparation of CSF solution

A solution of SF was fabricated as previously described [29, 30]. Briefly, raw cassava silk fibers were degummed thrice in boiling water with 0.05 wt% Na₂CO₃ at a bath ratio of 1:50 for 30 min, rinsed thoroughly, and dried at 60°C overnight. The extracted CSF fibers were dissolved in 9.3 mol × L⁻¹ LiBr solution by stirring at 60°C for 1.5 h. The regenerated CSF solution was then dialyzed in a cellulose tube against deionized water for 4 days to cut off the salt debris. The resulting solutions were centrifuged at 9,000 rpm for 20 min to remove the undissolved aggregates. The final concentrations of CSF solutions were adjusted to 3.5% (w/v) with distilled water.

2.3 Preparation of the CSF/HACC/GO composite films

A suitable amount of HACC was dissolved in 2% (v/v) acetic acid solution and magnetically stirred at room temperature overnight to prepare an HACC solution at 4 wt% with acetic acid. The diluted SF solution (2 wt%), glycerol (0.3 wt%), and glutaraldehyde (0.02 wt%) were incorporated into the HACC acetic acid solution (4 wt%) with acetic acid, respectively, dropwise. An amount of 100 mg GO powder was dispersed in 50 mL of distilled water by ultrasonication for 30 min, resulting in the fabrication of a suitable GO solution (2 mg × mL⁻¹). Then sodium hydroxide solution (1 mol × L⁻¹) was added to the GO solution to adjust its pH value to 10. This GO solution was introduced into the CSF/HACC solution dropwise, to form a symmetrical and transparent blend solution by stirring magnetically for 40 min. The final GO contents in the composite films were 0.2, 0.4, 0.6, and 0.8 wt% against the weight of CSF, respectively. The composite films were poured into a glass Petri dish and kept in an oven at 40°C for 48 h. Finally, they were rinsed with distilled water for 24 h to remove the residual glycerol and dried at room temperature.

2.4 Thermal properties of CSF/HACC/GO composite films

The thermal properties of the CSF/GO/HACC films were investigated by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA). TGA curves were obtained on a TA Q600 instrument at a heating rate of 10°C × min⁻¹under a dry nitrogen gas flow of 50 mL × min⁻¹. DSC was performed by a thermal analysis instrument (USA). The samples were heated from 0 to 600°C at 10°C×min⁻¹ under a nitrogen atmosphere.

2.5 Morphology and structure of CSF/HACC/GO composite films

The surface morphologies of the samples were studied with a field-emission scanning electron microscope (SEM; JEOL, Japan). X-ray diffraction (XRD) curves of the CSF/GO films were studied using a Mini flex II (Rigaku, Japan) diffractometer with Cu Ka radiation.

All the samples were slit into microparticles before analysis and assessed with 2q ranging from 5° to 50°. The formation of Fourier transform infrared (FT-IR) spectra (KBr pellet) on the films was studied on a Nicolet Magna-IR 750 spectrometer in the range of 4,000–500 cm⁻¹.

2.6 Behavior of CSF/HACC/GO composite films subjected to an enzyme reaction

The blend films were incubated at 37°C in 50 mL of PBS (the common laboratory buffer) solution with 1 U × mL⁻¹ protease-XIV for 5, 7, and 21 days. The enzyme solution was replenished with a fresh solution daily. The degradation rate was expressed as the percentage of weight loss relative to the initial dry weight. The residual samples were rinsed in distilled water and dried at 105° at constant weight for further characterization [31]. The degradation rate was presented as the percentage of weight loss relative to the initial dry weight.

3.1 Morphology

Figure 1 shows the surface and cross-sectional morphologies of three samples by SEM. Figure 1(A) exhibits a typical thin crumpled silk veil wavy morphology, while an interesting relatively smooth and flat white nanoglobular morphology is observed for the CSF/HACC film, as shown in Figure 1(B). With the incorporation of GO nanosheets, the surface of the CSF/HACC/GO composite film became rough, and a wrinkled structure of GO could be observed, as shown in Figure 1(C), indicating the optimum dispersion without aggregation and the excellent interactions between the three components in the films [28].

Figure 1

SEM images of the three samples: (A) surface of the GO, (B) surface of the CSF/HACC film, and (C) CSF/HACC/GO 0.8 wt% composite film.

3.2 Structural characteristics

The secondary structure of the CSF/HACC/GO composite films was determined by XRD and FT-IR. Figure 2(b–f) illustrates the XRD curves of the membranes. The characteristic XRD diffraction peak of CSF appeared at 11.8° and 22.0° which could be evidence of the a-helical structure; 16.5°, 20.2°, 24. 9°, 30.90°, 34.59° were attributed to the b-sheet structure. As shown in Figure 2(a), the characteristic XRD peak of GO appeared at 2q = 10.6°, corresponding to a layer-to-layer distance (d-spacing) of 0.83 nm [32]. Both the CSF/HACC sample and the composite samples displayed prominent peaks at 20.01° (Figure 2(b–f)), indicating a typical b-sheet structure. After GO was dispersed into the blend of CSF/HACC, the diffraction peak at 20.01° [33] was gradually enhanced as the GO content increased to 0.2 and 0.8 wt% (Figure 2(c–f)). Moreover, the diffraction peak of GO at 10.50° and 20.83° [33] was discovered in the CSF/HACC/GO composite films.

Figure 2

XRD curves of the CSF/HACC/GO films with different GO contents: (a) GO, (b) CSF/HACC, (c) CSF/HACC/GO 0.2 wt%, (d) CSF/HACC/GO 0.4 wt%, (e) CSF/HACC/GO 0.6 wt%, and (f) CSF/HACC/GO0.8 wt%.

Furthermore, Figure 3 illustrates the FT-IR spectra of the membranes. The infrared spectrum of CSF exhibited absorption band values of 1,640–1,615 cm⁻¹(amide I) attributable to the β-sheet structure, 3,291 cm⁻¹ assigned to the random coil conformation, and N–H stretching vibration of the protein molecule. Figure 3(a) illustrates the FT-IR spectra of GO, exceptional absorption bands at 1,727.69, 1,619.78, and 1,046.02 cm⁻¹, which were indicative of the C=O stretching vibration of the carboxylic group, C=C stretching vibration assigned to the sp² network of the unoxidized graphite domains, and C–O–C stretching vibration of the epoxy group. Figure 3(b) shows the characteristic spectrum of the CSF/HACC composite film and excellent absorption bands at 1,640.89 cm⁻¹was observed, demonstrating a typical β-sheet structure. Furthermore, the CSF/HACC composite film exhibited absorption bands at 1,063.02 and 1,149.82 cm⁻¹, which could be indicative of the characteristic vibration absorption peak of–OH in HACC and the stretching vibration of C–O–C bridge. Figure 3(c–f) shows the FT-IR spectra of the CSF/HACC/GO blend film. After GO was dispersed into the blend of CSF/HACC, the peaks recorded 1,230.17 cm⁻¹ (amide III β-sheet) a slight enhancement, implying that the molecular conformation of CSF transited from random coil/α-helix to β-sheet under the influence of GO. Meanwhile 1,063.02 cm⁻¹ (–OH absorption vibration) and 1,149.82 cm⁻¹ (C–O–C stretching vibration) also slightly increased. Furthermore, the typical characteristic peaks at 1,727.69 cm⁻¹ (C=O stretching vibration), 1,619.78 cm⁻¹ (C=C stretching vibration), and 1,046.02 cm⁻¹ (C–O–C stretching vibration of the epoxy group) did not appear in the FT-IR spectra of CSF/HACC/GO composite films. It was likely that HACC modified the surface of GO, and the latter was evenly dispersed in the composite film.

Figure 3

FT-IR curves of the CSF/HACC/GO films with different GO contents: (a) GO, (b) CSF/HACC, (c) CSF/HACC/GO 0.2 wt%, (d) CSF/HACC/GO 0.4 wt%, (e) CSF/HACC/GO 0.6 wt%, and (f) CSF/HACC/GO0.8 wt%.

3.3 Thermal properties

The thermal properties of the CSF/HACC/GO composite films were estimated by DSC and TGA. Figure 4 illustrates the TGA and DTG of the CSF/HACC sample and the composite samples. The TGA and DTG curves of the materials yielded three different features: the first was a weight loss which occurred at 60–100°C due to the evaporation of the bound water in the samples. Subsequently, the primary weight loss was from 190–300°C due to the thermal degradation of the volatile oxygen groups or the decomposition and melting of the amorphous zone of the internal structure of CSF with increase of temperature. The third weight loss was recorded above 300°C and this occurred due to the decomposition of more stable oxygen groups or the degradation of the crystallization portion with increasing temperature. When GO was dispersed in the composite films, both the CSF/HACC (Figure 4(b)) and the CSF/HACC/GO composite films (Figure 4(c–f)) exhibited homologous TGA and DTG curves. Figure 4(c–f) shows that the decomposition temperature improved with the increase of GO content. Especially when the content of GO was 0.8%, the degradation temperature ran up to 287.86°C, illustrating the higher thermal stability because of the strong intermolecular forces between the CSF or HACC molecular chains and the GO nanosheets compared with the CSF/HACC composite film.

Figure 4

(M) TG and (N) DTG curves of the SF/HACC/GO films with different GO contents: (a) GO, (b) CSF/HACC, (c) CSF/HACC/GO 0.2 wt%, (d) CSF/HACC/GO 0.4 wt%, (e) CSF/HACC/GO 0.6 wt%, and (f) CSF/HACC/GO0.8 wt%.

Furthermore, Figure 5 shows the DSC curves of the CSF/HACC sample and the composite samples. All the samples yielded an endothermic peak at around 128°C due to the evaporation of the adsorbed water molecules in the samples. The primary degradation peaks were produced in the region of 290–320°C between the CSF/HACC film (Figure 5(b)) and CSF/HACC/GO composite films (Figure 5(c–f)). With the incorporation of GO, Figure 5(c–f) shows that the decomposition temperature improved due to increase in the GO content compared with CSF/HACC composite film. When the content of GO was 0.8%, the degradation temperature reached 305.27°, implying the increase in content of the β-sheet crystal structure [34] because of the incorporation of GO.

Figure 5

DSC curves of the CSF/HACC/GO films with different GO contents: (a) GO, (b) CSF/HACC, (c) CSF/HACC/GO 0.2 wt%, (d) CSF/HACC/GO 0.4 wt%, (e) CSF/HACC/GO 0.6 wt%, and (f) CSF/HACC/GO0.8 wt%.

3.4 Mechanical properties

The mechanical properties of the SF-based composite film solutions were studied as previously described. GO was integrated into the CSF/HACC matrix to recompense for the mechanical properties [35], and to ensure that the fabricated CSF/HACC/GO-based biomaterial has sufficient mechanical properties as well as impressive toughness. The stress–strain curves of the SF films are shown in Figure 6, and the breaking strength and elongation at break are shown in Table 1. It could be observed that the breaking strength and elongation of the CSF/HACC film calculated were 40.24 ± 2.96 and 15.63 ± 3.27 MPa, respectively. With the increase of the amount of GO into the composite films, the breaking strength and elongation gradually improved, the value arrived at 97.69 ± 3.69 and 79.11 ± 1.48 MPa, respectively, at 0.8 wt% GO concentration. It was likely that some hydrogen bonds resulted between the CSF or HACC and GO.

Figure 6

Stress–strain curves of the CSF/HACC/GO films with different GO contents: (a) CSF/HACC, (b) CSF/HACC/GO 0.2 wt%, (c) CSF/HACC/GO 0.4 wt%, (d) CSF/HACC/GO 0.6 wt%, and (e) CSF/HACC/GO0.8 wt%.

3.5 Enzymatic degradation behavior of the SF/HACC/GO blend films

The degradation behavior [36,37,38] was very significant in the biomaterial field. The degradation behavior of SF membrane was related to its secondary structure. In the degradation atmosphere of protease, the degradation rate decreased gradually with the increased content of the b-sheet structure. In this study, CSF/HACC/GO blend films were fabricated to examine the effect of the blending ratio on the degradation behavior. Figure 7 shows the weight loss of the CSF/HACC/GO blend films during a degradation trend over 21 days by protease-XIV. It was found that the CSF/HACC film rapidly degraded, and the residual mass was only 51.79 ± 2.5% of the primary mass after 21 days. When GO was added to the composite films, the weight loss of the composite films decreased gradually with time and the degradation rate was decreased with the increase of the amount of GO. Especially when the content of GO was 0.8%, the residual mass was only 64.35 ± 1.1% of the primary mass after 21 days (Figure 7(e)). The incorporation of GO probably played an important role in enhancing the increase in the amount of the b-sheet crystal structure.

Figure 7

Enzymatic degradation behavior of the CSF/HACC/GO films with different GO contents: (a) CSF/HACC, (b) CSF/HACC/GO 0.2 wt%, (c) CSF/HACC/GO 0.4 wt%, (d) CSF/HACC/GO 0.6 wt%, and (e) CSF/HACC/GO0.8 wt%.

Furthermore, to exhibit the biodegradation behavior of the CSF/HACC)/GO blend films, Figure 8 illustrates the SEM images of CSF/HACC/GO films after incubation for 5, 7, and 21 days in protease-XIV solution. It was observed that the surface of the CSF/HACC film was relatively smooth except for some white particles with uniform sizes (Figure 8(a)). With incubation in enzyme solution, prominent surface erosion was found on the surface of the CSF/HACC film after 5 days of degradation (Figure 8(A-1)), and the degree of surface corrosion continued to be severe with increase in the incubation time (Figure 8(A-2 and A-3)). Also, numerous debris were found within 21 days of incubation with enzyme solution (Figure 8(A-3)) compared with the degradation time of 7 days (Figure 8(A-2)). With the incorporation of GO, the degree of surface erosion decreased with increase in the incubation time (Figure 8(B-1–B-3)), indicating the formation of more b-sheet crystal structures. It might also be that chemical crosslinking of regenerated CSF-based composite films played an important role in the biodegradability, including the degradation rate and degradation time of silk biomaterials. The CSF/HACC/GO composite membrane with 0.8 wt% GO concentration presented hole-shaped residuals both within 5 days of degradation (Figure 8(B-1)). And within 7 days (Figure 8(B-2)) or 21 days (Figure 8(B-3)) of incubation with enzyme solution (Figure 8(B-2)), the films were still able to maintain the integrity of the membrane structure. All these implied that the molecular conformation of CSF changed from random coil/a-helix to b-sheet, suggestive of the GO component importantly improving with an impressive degradability of the blend films.

Figure 8

SEM images of CSF/HACC/GO films after incubation for 5, 7, and 21 days in protease-XIV solution. (A-1–A-3) the CSF/HACC films were degraded for 5, 7, and 21 days in protease-XIV solution, (B-1–B-3) the CSF/HACC/GO films were degraded for 5, 7, and 21 days in protease-XIV solution. (A) SEM images of the CSF/HACC films before degradation and (B) SEM images of the CSF/HACC/GO films before degradation.