Endocytic Mechanisms of Graphene Oxide Nanosheets in Osteoblasts, Hepatocytes and Macrophages Javier Linares, † M. Concepció n Matesanz, † Mercedes Vila, ‡,§,⊥ M. Jose ́ Feito, † Gil Gonc ̧ alves, ⊥ María Vallet-Regí, ‡,§ Paula A. A. P. Marques, ⊥ and M. Teresa Portole ́ s* ,† † Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense, 28040 Madrid, Spain ‡ Department of Inorganic and Bioinorganic Chemistry, Faculty of Pharmacy, Instituto de Investigació n Sanitaria Hospital 12 de Octubre i+12, Universidad Complutense, 28040 Madrid, Spain § Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, 50018 Aragon, Spain ⊥ TEMA-NRD, Mechanical Engineering Department, University of Aveiro, 3810-193 Aveiro, Portugal ABSTRACT: Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC- PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC- PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications. KEYWORDS: endocytosis, graphene oxide, hepatocyte, macrophage, nanoparticle, osteoblast ■ INTRODUCTION Nano-graphene oxide (GO) and its functionalized derivatives have attracted great interest in nanomedicine due to its own intrinsic properties, all the dimensions lower than 100 nm and a singular chemical structure, 1 which allows interesting possible biomedical applications such as drug delivery, 2,3 tissue engineering 4,5 and hyperthermia cancer therapy. 6-8 The evaluation of the interactions at the interface of GO nanosheets and mammalian cells must be carried out before this nanomaterial can be used for biomedical applications. Concerning GO administration, although GO colloids are soluble in water, they need further functionalization with molecules like poly(ethylene glycol-amine) (PEG), which improves the material dispersion and stability in aqueous solutions. 2,9 Previous studies with PEGylated GO nanosheets labeled with fluorescein isothiocyanate (FITC-PEG-GOs) revealed differences in the amount of FITC-PEG-GOs uptake by Saos-2 osteoblasts, L929 fibroblasts, RAW-264.7 macro- phages and MC3T3-E1 preosteoblasts as a function of the cell type involved. Thus, FITC-PEG-GOs uptake was higher and faster in osteoblasts than in the other three cell types without inducing cell membrane damage. 10 Recent studies evidenced that for doses of 75 μg/mL, after cell internalization, FITC- PEG-GOs are preferentially localized on F-actin filaments, inducing cell-cycle alterations, apoptosis and oxidative stress in these cell types. 11 Concerning the potential use of nano- graphene oxide for hyperthermia cancer therapy, preliminary studies have shown effective induction of cell death in vitro after irradiating tumoral osteoblasts with internalized PEG- GOs. 6 However, the study of the toxicology of these nanosheets is still in its very early stages and it is necessary to know the entry mechanisms of PEG-GOs into mammalian cells for assessing its human toxicity. External nanoparticles can interact with the plasma membrane and enter into cells through different endocytosis processes. Two main types of endocytosis are distinguished depending on the size of the endocytic Received: May 21, 2014 Accepted: June 30, 2014 Published: June 30, 2014 Research Article www.acsami.org © 2014 American Chemical Society 13697 dx.doi.org/10.1021/am5031598 | ACS Appl. Mater. Interfaces 2014, 6, 13697-13706