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

Abstract

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.

Keywords::

• Uptake quantification
• macrophages
• fluorescent particles
• in vitro toxicity

Acknowledgments

The authors would like to acknowledge the help and support of their collaborators. They are grateful to Nano-H Company for providing the nanoparticles. They also acknowledge the excellent assistance of CTµ (Centre Technique des Microstructures) for cells samples preparation for TEM, CLYM (Centre Lyonnais de Microscopie) and CMES (Centre de Microscopie Electronique Stéphanois) for the access to the different electron microscopes. They are also grateful to Centre de Quantimétrie Cellulaire for the confocal images.

Declaration of interest

The authors would like to acknowledge the financial support of the Conseil Général de la Loire.