The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles.

Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein.

Role of physicochemical characteristics in the uptake of TiO2 nanoparticles by fibroblasts.

The relation between the physico-chemical properties of nanoparticles (NPs) and the degree of cellular uptake is incompletely elucidated. In this study, we investigated the influence on the cellular uptake of a wide range of fully characterized TiO2 NPs. L929 fibroblasts were exposed for 24 h to clinically relevant concentrations of nano-TiO2 and the degree of their association was assessed by ultrahigh resolution imaging microscopy (URI), scanning (SEM) and transmission (TEM) electron microscopy, as well as inductivity coupled plasma-mass spectroscopy (ICP-MS). The role of actin polymerization, a central feature of active internalization, was also studied and the results indicated that the internalization of TiO2 NPs involves a combination of actin-dependent uptake of large agglomerates as well as non actin-dependent uptake of small agglomerates. SEM and TEM revealed that the agglomerates of all NPs types were attached to the cellular membrane as well as internalized and confined inside cytoplasmic vesicles. URI and ICP-MS demonstrated that the particle association with cells was dose-dependent. The highest association was observed for spherical particles having mixed anatase-rutile crystallographic phase and the lowest for spindle-shaped rutile particles. ICP-MS revealed that the association was size-dependent in the order 5>10>40 nm for anatase spherical nanoparticles.

Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium.

The physicochemical characterization of nanoparticles in suspension is a prerequisite for the adequate assessment of their potential biological effect. Little is known to date about the colloidal stability of TiO2 nanoparticles in cell culture medium. This study investigates the effect of particle concentration, ionic strength, pH, and the presence of fetal bovine serum (FBS) and human serum albumin (HSA) on the colloidal stability of TiO2 nanoparticles in RPMI cell culture medium, by sedimentation measurements, dynamic light scattering, and electrokinetic measurements (zeta-potential). TEM revealed that the particles were polydisperse, with diameters ranging from approximately 15 to approximately 350 nm. The agglomeration rate and sedimentation rate increased with particles' concentration. The size of the agglomerates at 100 mg/L TiO2 was significantly reduced, from 1620+/-160 to 348+/-13 and 378+/-15 nm, upon the addition of 10% (v/v) FBS and 1% (w/w) HSA, respectively. The isoelectric point of TiO2 in water was 2.9 and the measured zeta-potential in RPMI was -16+/-2 mV at pH 7.4. A slight increase in the zeta-potential of TiO2 in RPMI was observed upon the addition of FBS and HSA. The addition of FBS and HSA prevented high agglomeration, leading to a stable dispersion of TiO2 nanoparticles for at least 24 h, possibly due to steric stabilization of the particles.