Analysis of nanoparticle biomolecule complexes.

Nanoparticles exposed to biological fluids adsorb biomolecules on their surface forming a biomolecular corona. This corona determines, on a molecular level, the interactions and impact the newly formed complex has on cells and organisms. The corona formation as well as the physiological and toxicological relevance are commonly investigated. However, an acknowledged but rarely addressed problem in many fields of nanobiotechnology is aggregation and broadened size distribution of nanoparticles following their interactions with the molecules of biological fluids. In blood serum, TiO2 nanoparticles form complexes with a size distribution from 30 nm to more than 500 nm. In this study we have separated these complexes, with good resolution, using preparative centrifugation in a sucrose gradient. Two main apparent size populations were obtained, a fast sedimenting population of complexes that formed a pellet in the preparative centrifugation tube, and a slow sedimenting complex population still suspended in the gradient after centrifugation. Concentration and surface area dependent differences are found in the biomolecular corona between the slow and fast sedimenting fractions. There are more immunoglobulins, lipid binding proteins, and lipid-rich complexes at higher serum concentrations. Sedimentation rate and the biomolecular corona are important factors for evaluating any experiment including nanoparticle exposure. Our results show that traditional description of nanoparticles in biological fluids is an oversimplification and that more thorough characterisations are needed.