Bioavailability of fullerene nanoparticles : factors affecting membrane partitioning and cellular uptake

Interactions of engineered nanomaterials (ENMs) with environmental interfaces have become a critical aspect of environmental health and safety evaluations. Carbon fullerene (C₆₀) has emerged at the forefront of nanoscale research and applications due to its unique properties. Although there are concerns associated with the harmful effects of fullerene towards living organisms, the mechanisms of fullerene toxicity are still under debate. A first step toward assessing these mechanisms requires evaluation of the bio-accumulation and bio-uptake of fullerene through lipid membranes which serve as biological barriers in cells. In this dissertation, partitioning of fullerene between water and lipid membranes and cellular uptake of fullerene were investigated to assess bioavailability of this nanoparticle. Traditional methods to estimate the equilibrium partitioning of molecular level chemicals between water and lipid membranes (K[subscript lipw]) cannot be applied to measure K[subscript lipw] of nanoparticles due to the large size of nanoparticle aggregates. In this study, we developed an in vitro method to estimate K[subscript lipw] of fullerene using solid supported lipid membranes (SSLMs) with various membrane compositions. K[subscript lipw] of fullerene increased with increasing acyl chain length and K[subscript lipw] values were higher after creating phase separation in ternary lipid membranes compared to pre-phase separation. In addition, the partitioning values (K[subscript lipw]) were found to depend on the lipid head charges. These results suggest that the lipid membrane composition can be a critical factor for assessing bioaccumulation of fullerene. Evaluation of the partitioning thermodynamics of fullerene demonstrated that the partitioning mechanism of fullerene is different from that of molecular level chemicals. It is generally acknowledged that molecular level chemicals partition into the hydrophobic center of lipid membranes (i.e., absorption), however, the partitioning mechanism of fullerene is a combination of adsorption on the lipid membrane surface and absorption. Caco-2 cellular uptake of fullerene nanoparticles was investigated using an in vitro method developed in this study to distinguish between active and passive transport across cell membranes. Energy dependent endocytosis is hypothesized to be the main cellular transport mechanism based on an observed temperature dependence of cellular uptake and evidence for saturation of the active sites of transport during cellular uptake of fullerene. Metabolic inhibitors decreased the mass of fullerene taken up by the cells, which supports an active transport mechanism of fullerene through the cell membranes. To evaluate bioavilability of fullerene under environmentally relevant conditions, the effects of humic acid and fetal bovine serum (FBS) on the lipid accumulation and cellular uptake were also investigated. Humic acid and FBS changed the surface characteristics of fullerene. The presence of FBS significantly decreased lipid accumulation of fullerene presumably due to higher steric hinderance of FBS coated fullerene as well as the changes in surface energy, water solubility, and lipid solubility of charged FBS coated fullerene relative to that of bare fullerene. Both humic acid and FBS also effectively lowered the cellular uptake of fullerene. These results imply that natural organic matter and biomolecules in natural aquatic and biological environments have significant effects on the bioavilability of fullerene nanoparticles