Graphene-based nanomaterials (GBNs) due to their exceptional mechanical, electronic, and thermal properties assumed significance in a variety of applications. The cytotoxic properties of GBNs are also important for their biomedical applications. GBNs have been shown to be cytotoxic toward a variety of cell types. However, the impact of alignment of nanosheets on the antibacterial activity has not been established. Researchers at Department of Chemical and Environmental Engineering, Yale University, USA, have investigated orientation-dependent interaction of graphene oxide (GO) nanosheets aligned in different orientations using a magnetic filed with Escherichia coli (E. coli). The GO nanosheets with vertical orientation exhibit an enhanced antibacterial activity when compared to those with random and horizontal orientations and the mechanism responsible is also suggested.
The schematic illustration of alignment of GO nanosheets with different orientations using magnetic field and alignment quality of GO nanosheets suspended in the monomer solution at different field strengths evaluated by 2D small-angle X-ray scattering (SAXS) are shown in Fig. 1.
Fig. 1 (a) Schematic illustration of alignment of GO nanosheets with different orientations using magnetic field; and (b) alignment quality of GO nanosheets
The various stages involved in the fabrication of GO composite films is shown in Fig. 2. Suspensions of GO nanosheets (with a thickness of ∼0.8 nm) in 2-hydroxyethyl methacrylate (HEMA), doped with cross-linker and photo initiator, were sealed between two glass substrates with a 300-μm spacer and aligned in a magnetic field of 6 T. Samples were subsequently cross-linked under UV irradiation to form polymer films, which preserved the orientation of the aligned GO nanosheets. The composite films were then detached from the glass substrates and irradiated using UV/O3 to etch away the outer polymer and expose GO nanosheets on the surface. The resultant films are tough, mechanically coherent and resistant to water swelling, which are critical in preserving the GO orientation in aqueous environments.
Fig. 2 Various stages involved in the fabrication of GO composite film
The GO composite films were contacted with E. coli in suspension for 3 h. The bacteria attached on the surface were stained using SYTO 9 dye and propidium iodide and evaluated for live and dead cells. The vertical-GO film showed a lower cell viability (56.0 ± 8.7%) when compared to those with random (75.3 ± 3.5%) and planar (81.8 ± 5.1%) orientation. Morphological features indicate that E. coli on No-GO film showed an intact cell morphology, indicating no cytotoxicity of the pure polymer. E. coli on planar- and random-GO films largely retained their morphological integrity whereas cells on vertical-GO films became flattened and wrinkled, suggesting loss of viability and possible damage to the cell membrane (Fig. 3).
Fig. 3 SEM micrographs of E. coli cells on etched GO composite films. The scale bar is 1 μm.
The mechanism for the enhanced antibacterial activity of vertically aligned GO nanosheets is explained based on (i) physical disruption; and (ii) chemical oxidation using lipid vesicles and oxidation of glutathione, respectively. GO nanosheets with a vertical orientation induced physical disruption of the lipid bilayer structure, resulting in loss of membrane integrity of the GO/lipid vesicle system. GO nanosheets with a vertical orientation also increased the extent of oxidation of glutathione (27.6%) with limited generation of reactive oxygen species, suggesting that the oxidation occurs through a direct electron transfer mechanism. Thus, both mechanisms contribute to the enhanced antibacterial activity of the vertical-GO film. Nevertheless, both of them require direct, edge-mediated contact with cells. The exposed edges of GO nanosheets with a vertical orientation could induce enhanced physical penetration and promote greater levels of electron transfer. Hence, the enhanced antibacterial activity of the film with vertically aligned GO nanosheets can be attributed to the increased density of edges with a preferential orientation for membrane disruption. The orientation-dependent cytotoxicity of GO nanosheets has direct implications on the design of engineering surfaces using graphene based nanomaterials.
T.S.N. Sankara Narayanan
For more information, the reader may kindly refer: X. Lu et al., Enhanced antibacterial activity through the controlled alignment of graphene oxide nanosheets, PNAS 2017 114: E9793-E9801
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