Biodistribution and acute toxicity of a nanofluid containing manganese iron oxide nanoparticles produced by a mechanochemical process

Abstract

Mariangela Bellusci,1 Aurelio La Barbera,1 Franco Padella,1 Mariateresa Mancuso,2 Alessandra Pasquo,2 Maria Giuseppa Grollino,2 Giorgio Leter,2 Elisa Nardi,3 Carlo Cremisini,3 Paola Giardullo,4 Francesca Pacchierotti21Technical Unit for Material Technologies, 2Technical Unit for Radiation Biology and Human Health, 3Technical Unit for Environmental Characterization, Prevention and Recovery, Agenzia Nazionale per le Nuove Tecnologie, l'Energia e lo Sviluppo Economico Sostenibile (ENEA), Casaccia Research Centre, Rome, Italy; 4Department of Radiation Physics, Marconi University, Rome, ItalyAbstract: Superparamagnetic iron oxide nanoparticles are candidate contrast agents for magnetic resonance imaging and targeted drug delivery. Biodistribution and toxicity assessment are critical for the development of nanoparticle-based drugs, because of nanoparticle-enhanced biological reactivity. Here, we investigated the uptake, in vivo biodistribution, and in vitro and in vivo potential toxicity of manganese ferrite (MnFe2O4) nanoparticles, synthesized by an original high-yield, low-cost mechanochemical process. Cultures of murine Balb/3T3 fibroblasts were exposed for 24, 48, or 72 hours to increasing ferrofluid concentrations. Nanoparticle cellular uptake was assessed by flow-cytometry scatter-light measurements and microscopy imaging after Prussian blue staining; cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony-forming assays. After a single intravenous injection, in vivo nanoparticle biodistribution and clearance were evaluated in mice by Mn spectrophotometric determination and Prussian blue staining in the liver, kidneys, spleen, and brain at different posttreatment times up to 21 days. The same organs were analyzed for any possible histopathological change. The in vitro study demonstrated dose-dependent nanoparticle uptake and statistically significant cytotoxic effects from a concentration of 50 µg/mL for the MTT assay and 20 µg/mL for the colony-forming assay. Significant increases in Mn concentrations were detected in all analyzed organs, peaking at 6 hours after injection and then gradually declining. Clearance appeared complete at 7 days in the kidneys, spleen, and brain, whereas in the liver Mn levels remained statistically higher than in vehicle-treated mice up to 3 weeks postinjection. No evidence of irreversible histopathological damage to any of the tested organs was observed. A comparison of the lowest in vitro toxic concentration with the intravenously injected dose and the administered dose of other ferrofluid drugs currently in clinical practice suggests that there might be sufficient safety margins for further development of our formulation.Keywords: biodistribution, biosafety, ICP-MS, Prussian blue staining, MnFe2O4 nanoparticles, ferrofluid