Water-Soluble Fullerene (C(60)) Derivatives as Nonviral Gene-Delivery Vectors

[Image: see text] A new class of water-soluble C(60) transfecting agents has been prepared using Hirsch−Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C(60) vectors in gene delivery and expression, several different C(60) derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C(60) derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C(60) derivatives, namely, an octa-amino derivatized C(60) and a dodeca-amino derivatized C(60) vector. All C(60) vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C(60) vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C(60) compounds, the C(60) vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C(60)-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C(60) vectors suggests the possibility of developing analogous Gd@C(60)-based vectors to serve simultaneously as both therapeutic and diagnostic agents.