Molecular control of interfacial protein structure on graphene-based substrates steers cell fate

Abstract

The use of graphene-based materials (GBMs) for tissue-engineering applications is growing exponentially due to the seemingly endless multi-functional and tunable physicochemical properties of graphene, which can be exploited to influence cellular behaviours. Despite many demonstrations wherein cell physiology can be modulated on GBMs, a clear mechanism connecting the different physicochemical properties of different GBMs to cell fate has remained elusive. In this work, we demonstrate how different GBMs can be used to cell fate in a multi-scale studystarting from serum protein (Fibronectin) adsorption to molecular scale morphology, structure and bioactivity, and finally ending with stem cell response. By changing the surface chemistry of graphene substrates with only heating, we show that molecular conformation and morphology of surface adsorbed fibronectin controls epitope presentation, integrin binding, and stem cell attachment. Moreover, this subtle change in protein structure is found to drive increased bone differentiation of cells, suggesting that physicochemical properties of graphene substrates exert cell control by influencing adsorbed protein structure.Keywords: Graphene-based materials (GBMs), protein interaction, protein molecular structure, protein bioactivity, stem cell differentiation, identification of biochemical activity changes of insulin on graphene and GO surfaces was presented. Experiments directly probing protein molecular structure, conformation, and exposure of bioactive domains of proteins are required to unequivocally demonstrate if GBMs modulate protein structure to influence stem cell interaction. In addition, controlled experiments evaluating the effect of the adsorbed protein and its molecular features on stem cell attachment and differentiation should be done in absence and presence of serum. Providing such experiments will provide mechanistic insight on GBM-mediated stem cell response for future rational design of materials from graphene.Here, we study ECM protein adsorption, interaction, orientation and structure from the macroscale to the molecular scale on GBMs and perform stem cell culture in the presence and absence of serum to relate the physicochemistry of GBMs with protein structure and cell fate. We used GO and RGOthe two most commonly used GBMs as exemplary engineering materialsto evaluate bioactivity of GBMs having different surface chemistries by investigating the effect of GBM chemistry on fibronectin (FNa protein abundant in the ECM), adsorption, orientation, and structure. We couple these surface characterization experiments with biochemical activity assays and stem cell response experiments, allowing us to elucidate the link between physicochemical properties of GBMs, FN protein unfolding, altered biochemical activity, and stem cell differentiation.Corresponding Authors

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“…And due to the high electronegativity of fluorine, C-F bonds can induce biological responses to promote cell adhesion and proliferation [22,23]. (3) High surface area and drug loading capacity [24,25]. Aromatic structure and fluorine of FG allow it to form strong π-π and hydrogen bond interactions with drugs, which makes it possible to absorb GOx and form a stable delivery system [26,27].…”