Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

BACKGROUND: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. METHODS: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). RESULTS AND CONCLUSION: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms.

BACKGROUND: The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments. METHODS: Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production]. RESULTS AND CONCLUSION: Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49 ± 11%) than for amine 1-µm beads (18 ± 5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446 ± 7 and 139 ± 12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H(2)O(2) production.