Graphene Oxide Strengthens Gelatine through Non-Covalent Interactions with Its Amorphous Region

Abstract

Graphene oxide (GO) has attracted huge attention in biomedical sciences due to its outstanding properties and potential applications. In this study, we synthesized GO using our recently developed 1-pyrenebutyric acid-assisted method and assessed how the GO as a filler influences the mechanical properties of GO-gelatine nanocomposite dry films as well as the cytotoxicity of HEK-293 cells grown on the GO-gelatine substrates. We show that the addition of GO (0-2%) improves the mechanical properties of gelatine in a concentration-dependent manner. The presence of 2 wt% GO increased the tensile strength, elasticity, ductility, and toughness of the gelatine films by about 3.1-, 2.5-, 2-, and 8-fold, respectively. Cell viability, apoptosis, and necrosis analyses showed no cytotoxicity from GO. Furthermore, we performed circular dichroism, X-ray diffraction, Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy analyses to decipher the interactions between GO and gelatine. The results show, for the first time, that GO enhances the mechanical properties of gelatine by forming non-covalent intermolecular interactions with gelatine at its amorphous or disordered regions. We believe that our findings will provide new insight and help pave the way for potential and wide applications of GO in tissue engineering and regenerative biomedicine.

Keywords: 2D nanomaterials; biomaterials; cytotoxicity; gelatine; graphene oxide; mechanical property of gelatine.

Figure 1

Characterizations of GO used in this study. (A,B) AFM image and corresponding height profiles (C) Raman spectrum; (D) XPS survey spectrum. More detailed information can be found in Table 1 and our previous publications [57,58].

Figure 2

Mechanical properties of gelatine, GO, and GO–gelatine nanocomposite films. (A) Stress–strain tensile behaviour profiles with the color representations indicated. (B–E) Correlations between GO (wt%) and the tensile strength (B), elastic modulus (C), ductility (D), and relative energy absorption (E). The error bars represent the standard error of the mean (SEM) of three independent experiments (n = 3) for all samples.

Figure 3

Assessment of cell viability on substrates coated with gelatine, GO, and various GO–gelatine composites. (A) Cross-sectional schematic diagram of the experimental set-up in a single well of a 24-well plate. (B) Cell viability based on Alamar blue assay after the cells were grown in different substrates for 5 days. (C,D) Time courses of RealTime-Glo^TM phosphatidylserine-annexin V apoptosis (C, Luminescence) and necrosis (D, Fluorescence) assays. The solid lines represent pseudo-first-order association kinetics. The error bars represent the SEM of at least three independent experiments (n ≥ 3) for all samples. One-way ANOVA showed no statistically significant difference in all cases, p-values > 0.05.

Figure 4

Physical and chemical characterizations of GO–gelatine composites. (A) Far UV CD spectra of gelatine solution in the absence and presence of 1 wt% GO. (B) XRD pattern; (C) FTIR spectra; and (D) XPS survey spectra of various GO–gelatine composite films. The color representations are the same in (B–D), as indicated in the plots.

Figure 5

Schematic diagram of how GO improves mechanical property of GO–gelatine nanocomposite. Basic chemical structures of GO and gelatine are presented on top row. The proposed GO–gelatine composite interactions are shown in the bottom row, with examples of intermolecular hydrogen bonding interactions (black dash lines) formed between GO (red) and gelatine chain at amorphous or disordered ranges (blue) dispersed on the right. The triple-helix structure of gelatine is shown in three colors.