Characterization of fullerene colloidal suspension in a cell culture medium for in vitro toxicity assessment

Abstract

To elucidate important parameters for in vitro toxicity assessment of C₆₀ and C₇₀ fullerene colloidal particles, experiments were carried out in culture medium using pulsed field gradient nuclear magnetic resonance (PFG-NMR), asymmetrical flow field-flow fractionation (AFFFF), and dynamic light scattering (DLS) methods. First, the amounts of total and bulk bovine serum albumin (BSA) molecules in C₆₀ and C₇₀ fullerene colloidal suspensions were determined using the PFG-NMR and AFFFF methods. Because the amount of bulk BSA molecules in the cell culture medium is a significant factor in inducing cell growth and because BSA can strongly adsorb onto the fullerene particles, this value is an important parameter for toxicological assessment. It was found that most of the BSA molecules are freely diffusing for both fullerene colloidal suspensions, at least in the range of fullerene concentration from 0.0025–0.15 mg mL⁻¹. Second, structural analysis of the fullerene colloidal nanoparticles was successfully performed using AFFFF-multi angle light scattering (MALS) and DLS methods. Based on the observed light scattering profiles obtained from a narrow size distribution of colloidal particles collected after AFFFF separation, it was estimated that the fullerene colloidal nanoparticles of both C₆₀ and C₇₀ did not adopt a hard spherical structure in the culture medium. The results from combined analysis using the AFFFF-MALS and DLS methods also supported this conclusion and indicated that the fullerene colloidal particles adopted a more flexible structure in culture medium. Since carbon nanomaterials with different geometric structures exhibit quite different cytotoxicity and bioactivity, these results are important for in vitro toxicity assessment.