Toxicological Evaluation Of Medical Janus Fe₃O₄-TiO₂ Nanoparticles

Objective: To evaluate the toxicities of this newly synthesized Janus Fe₃O₄-TiO₂ nanoparticles（NPs）in vitro and in vivo,for providing scientific basis for the safe use of Fe₃O₄-TiO₂ NPs in cancer diagnosis and treatment.Methods: For comparison,its maternal material TiO₂ NPs was also evaluated.To evaluate the toxicity and underlying molecular mechanisms of the Janus Fe₃O₄-TiO₂ NPs,an in vitro study in a human liver cell line HL7702 cells was conducted.In vitro,MTT experiment was used to compare the effect of Fe₃O₄-TiO₂ NPs and TiO₂ NPs on the viability of HL7702 cells.Mitochondrial membrane potential detection,ATP detection,MDA detection,ROS detection and apoptosis analysis were conducted to investigate the underlying mechanisms of the Janus Fe₃O₄-TiO₂ NPs and its maternal material TiO₂ NPs.In vivo,rats were treated with tail intravenous injections with a single dose of Janus Fe₃O₄-TiO₂ NPs or TiO₂ NPs in saline.Then organ weight/body weight coefficients,hematological,biochemical,histopathological as well as molecular protein expressions were analyzed after 30 days treatment.The inductively coupled plasma mass spectrometry（ICP-MS）was used for the determination of Fe and Ti element content in the tissue samples of heart,liver,lungs,spleen,kidneys,brain and testes and the accumulation of Fe₃O₄-TiO₂ NPs in the animal bodies.Results: The average size distribution of the Fe₃O₄-TiO₂ NPs is about 20-25 nm and the TiO₂ NPs is about 7-10 nm.Results in the culture cells showed that both Fe₃O₄-TiO₂ NPs and TiO₂ NPs treatment decreased cell viability and ATP levels,but malonaldehyde（MDA）and reactive oxygen species（ROS）generation increased.Mitochondria JC-1 staining assay showed that mitochondrial membrane permeability injury occurred in both NPs treated cells.Cell viability analysis showed that TiO₂ NPs induced a little higher cytotoxicity than Fe₃O₄-TiO₂ NPs in HL7702 cells.Western blot indicates that both TiO₂ NPs and Fe₃O₄-TiO₂ NPs could induce apoptosis,inflammation,as well as carcinogenesis related signal protein alterations.Fe₃O₄-TiO₂ NPs increased protein expressions of p-Erk,p-JNK,p-p38,p53,c-fos,COX-2,p-NF-κB,NQO1,Fas,Cytochrome c,Cleaved Caspase 9,Cleaved Caspase 3 and Metallothionein（MT）.TiO₂ NPs increased protein expressions of p-Erk,p-JNK,c-Jun,NQO1,R-Ras,HO-1 and MT.These results indicate that with the treatment dose increase,both Fe₃O₄-TiO₂ NPs and TiO₂ NPs induce oxidative damage and carcinogenesis related signal protein alterations in human liver cells.Comparatively,Fe₃O₄-TiO₂ NPs induce higher signal protein expressions than TiO₂ NPs under a high treatment dose.However,under a low dose（6.25 μg/cm2）,neither NPs had any significant toxicity on human liver cells.The results in vivo showed that,only a slight Ti element accumulation in the heart,spleen and liver could be found in the Janus Fe₃O₄-TiO₂ NPs groups（P>0.05,compared with control）.However,significant Ti element accumulation in the spleen,lungs and liver was found in the TiO₂ NPs treated rats.Both Fe₃O₄-TiO₂ NPs and TiO₂ NPs could induce certain histopathological abnormalities.Western-blot analysis showed that both Fe₃O₄-TiO₂ NPs and TiO₂ NPs could induce certain apoptotic and inflammatory related molecular protein up-regulations in rat livers.A certain degree of alteration in liver function,electrolyte and lipid parameters was also observed in both material treated rats.Compared to the Janus structure Fe₃O₄-TiO₂ NPs treated groups,TiO₂ NPs at 30 mg/kg showed more sever adverse effects in the animals.Conclusions: Under a low dose,neither NPs showed significant toxicities on human liver cells and treated animals.But at high treatment doses,both Fe₃O₄-TiO₂ NPs and TiO₂ NPs could induce oxidative stress and have a potential carcinogenetic effect in vitro,and both materials could induce certain degree of damages in lungs,spleen,liver and kidneys in animals.Janus Fe₃O₄-TiO₂ NPs seems certain less toxic than TiO₂ NPs in rats at 30 mg/kg.Although toxicities of Fe₃O₄-TiO₂ NPs does not increase compared to TiO₂ NPs,to ensure safe use of this newly developed Janus NPs in cancer diagnosis and therapy in the future,further studies are needed for elaborating the detail mechanisms of a high dose of Fe₃O₄-TiO₂ NPs induced toxicity.In addition,chronic animal studies are also needed to evaluate their long-term bio-effects.