Endocytic pathways involved in polystyrene nanoparticle uptake in human gastric adenocarcinoma cells

Nanoparticles (NPs) are promising tools in medical fields, both in diagnosis and therapy (Schlorf et al., 2012; Wickline and Lanza, 2008). Despite this high applicative potential, little is known about their interaction with biological systems, almost in terms of endocytic pathways and toxicity. The first step to develop a good drug delivery systems based on NPs is to well characterize these molecular aspects. Thus, in this work, with a quantitative and qualitative approach, we studied the uptake of two representative sizes of polystyrene nanoparticles (PS-NPs), 44 nm (NP44) and 100 nm (NP100), labeled with FITC and ROD, respectively, in human adenocarcinoma gastric cells (AGS). The experiments were performed after exposure with 10μg/mL NPs for different times of incubation and temperatures (37°C and 4°C), with or without well known endocytosis inhibitor drugs (dynasore for clathrin dependent pathways and EIPA for macropinocytosis/phagocytosis). Quantitative spectrofluorimetric assays reveal a time-dependent kinetics of internalization at 37°C, with maximum values after 30 min and a decrease after 1 h for both NPs sizes. Precisely, NP44 show a high rate of uptake and a quickly internalization compared to NP100 (Fig. 1). Fluorescent images demonstrate that NPs are able to accumulate in the cytoplasm after 1 and 4 h, without reaching cell nuclei. However, NP100 tend to form aggregate after long exposition times (Fig. 3), while NP44 present an uniform cytoplasmatic distribution at all times considered (Fig. 4). Endocytosis inhibition tests show a null internalization at 4° C and a strong reduction of the uptake rate after treatment with dynasore for both NPs; EIPA, instead, partially affects NPs uptake (Fig. 2). In conclusion, in this study, we demonstrated that PS-NPs are internalized by AGS cells in a size and time dependent manner; probably, as suggest by other authors, they undergo a release process (Iversen et al., 2011). Moreover, we show that this uptake occurs through an energy dependent mechanism and that clathrin mediated endocytosis seems to be the privileged endocytic pathway for PS-NPs. References: Iversen TG., SkotlandT. Sandvig K. (2011). Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nonotoday 6:176-181. Schlorf T, Meincke M, Kossel E, Gluer CC, Jansen O, Mentlein R (2011). Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging. Int J Mol Sci. 12(1):12–23. Wickline S.A., Lanza G.M. (2003). Nanotechnology for molecular imaging and targeted therapy. Circulation 107: 1092–1095.