Plasma membrane-associated graphene oxide as aplatform for modulating signalling through cell-surfacereceptors: an integrin-focused proof-of-concept study

Abstract

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.