Covalent functionalization of graphene sheets for plasmid DNA delivery: experimental and theoretical study

Several approaches, including plasmid transfection and viral vectors, were used to deliver genes into cells for therapeutic and experimental purposes. However, due to the limited efficacy and questionable safety issues, researchers are looking for better new approaches. Over the past decade, graphene has attracted tremendous attention in versatile medical applications, including gene delivery, which could be safer than the traditional viral vectors. This work aims to covalently functionalize pristine graphene sheets with a polyamine to allow the loading of plasmid DNA (pDNA) and enhance its delivery into cells. Graphene sheets were successfully covalently functionalized with a derivative of tetraethylene glycol connected to polyamine groups to improve their water dispersibility and capacity to interact with the pDNA. The improved dispersibility of the graphene sheets was demonstrated visually and by transmission electron microscopy. Also, it was shown by thermogravimetric analysis that the degree of functionalization was about 58%. Moreover, the surface charge of the functionalized graphene was +29 mV as confirmed by zeta potential analysis. The complexion of f-graphene with pDNA was achieved at a relatively low mass ratio (10 : 1). The incubation of HeLa cells with f-graphene loaded with pDNA that encodes enhanced green fluorescence protein (eGFP) resulted in the detection of fluorescence signal in the cells within one hour. f-Graphene showed no toxic effect in vitro. Density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) calculations revealed strong binding with ΔH ₂₉₈ = 74.9 kJ mol^-1. QTAIM between the f-graphene and a simplified model of pDNA. Taken together, the developed functionalized graphene could be used for the development of a new non-viral gene delivery system.