D. Diaz-Diestra, B. Thapa, J. Carpena, F. Fisol, N. Konduru, J. Beltran-Huarac, G. Morell, B. Weiner
University of Puerto Rico Rio Piedras,
Keywords: Graphene Oxide, protein corona, toxicity
Summary:Diaz-Diestra, Daysi1,2,4, Thapa, Bibek1,3, Fisol, Faisalina,4,5 Carpena-Nunez, Jennifer,6 Konduru, Nagarjun,4 Beltran-Huarac, Juan,7 Morell, Gerardo 1,3 and Weiner, Brad R.1,2 1 Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA 2 Department of Chemistry, University of Puerto Rico, San Juan, PR 00936, USA 3 Department of Physics, University of Puerto Rico, San Juan, PR 00936, USA 4 Department of Environmental Health, Molecular and Integrative Physiological Sciences Program, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA 5 School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia 6 Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH 45433, USA 7 UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27514, USA Given the increasing incorporation of nanomaterials (NMs) into nano-enabled products and likely scenario of their release in to the environment, toxicological characterization of NMs is of paramount importance to protect health. The traditional toxicity testing is based on in vitro assays where NM suspensions are directly pipetted into the cell culture media and their incubation with cells over a time course. In order to prepare a stable and well dispersed NMs solutions, various sonication methods are used. It has been demonstrated that for 3D NMs these methods can introduce variability in the nanoparticle suspension properties e.g. agglomerate size, suspension stability, affecting their in vitro toxicity profiles. The impact of sonication methods in the case of 2D materials such as Graphene Oxide (GOx) has not been well studied. Here we investigated the effect of two widely used sonication methods, bath sonication and ultrasonication, on the dispersion stability and toxicity of GOx on the macrophage cell line RAW 264.7. The effective delivery energy sonication was measured and the GOx suspensions were carefully characterized to detect any chemical and structural changes by surface analytical techniques. The stability of GOx in cell culture media was also assessed along with the protein corona formation on GOx NPs. We found that more stable GOx dispersions were obtained by a discrete ultrasonication method and that these had a higher protein corona. This, in turn, translated into lower toxicity responses in macrophages as demonstrated by the metabolic and membrane cell assessment. This study clearly demonstrates the impact of dispersion methods on the in vitro toxicity of GOx and provides insights that at least clarify part of the contradictory results on recent GOX toxicity in-vitro studies.