Research On Toxicity Of Ultra-Small Fe₃O₄ Nanoparticles

In recent years,the rapid development of nanotechnology has led to the advancement of nanomedicine.More and more nanoparticles are being used in the fields of biosensing,simultaneous imaging,drug delivery and cancer diagnosis.Among these,Fe₃O₄ nanoparticles have attracted extensive interest,which have been found versatile in bio-applications such as cell separation,magnetic resonance imaging?MRI?,targeted drug delivery and photothermal therapy?PTT?due to their good biocompatibility,unique magnetic properties,and photothermal properties.It is generally believed that iron oxide has low toxicity and could be metabolized in vivo.However,most toxicity research were based on iron oxide nanoparticles with size above10 nm.Few studies on the toxicity of nanoparticles below 10 nm were carried out,while ultrasmall iron oxide nanoparticles presenting a good candidate for positive T1-weighted imaging,so research on its toxicity is necessary.In our research,we found that the ultrasmall superparamagnetic iron oxide nanoparticles?USPIONs?with size<5 nm were highly toxic and lethal at a dosage of 100 mg/kg.In contrast,no toxicity was found for USPIONs with size>5 nm.In this paper,the toxicology mechanism of USPIONs was studied,and the hypothesis of its toxicity mechanism is proposed and verified:First,ultrasmall nanoparticles?<5 nm?stimulate cells to generate reactive oxygen species?ROS?,and the ROS convert into hydrogen peroxide?H₂O₂?by the intracellular peroxidase;then,the generated H₂O₂ is catalyzed by Fe²⁺via Fenton reaction to produce hydroxyl radicals?·OH?,which are highly toxic and can't be scavenged by any known enzymes in vivo.Concrete study content as follows:1.The 2.3 nm,4.2 nm,and 9.3 nm Fe₃O₄ NPs were synthesized following a DEG-mediated modified solvothermal reaction,SiO₂ and Au NPs with similar particle size gradients were synthesized and used as controls.The characterization results of the synthesized nanoparticles showed that the prepared nanoparticles were nearly spherical in appearance,and have good dispersion.Then,we investigated the release of Fe²⁺and the Fenton reaction of Fe₃O₄ NPs.The results showd that the Fe²⁺released quicker from smaller Fe₃O₄ NPs due to the relatively higher specific surface,and this reaction is pH-dependent.In acidic medium,Fe₃O₄ NPs could catalyze the H₂O₂ to generate·OH via Fenton reaction,but the generated·OH was negligible at pH=7.4.2.Investigating the in vitro biological behavior of different sized nanoparticles.First,the cell uptake behavior of nanoparticles was investigated,and the results showed that for all kinds of nanoparticles,only<5 nm nanoparticles could enter the nuclei while those with a particle size>5 nm cannot.Then,we dectected the ROS and the types of ROS in cells induced by the different kinds of nanoparticles.The results showed that all nanoparticles with the size<5 nm could induce the production of O₂^-and H₂O₂ in cells,but only 2.3 nm and 4.2 nm Fe₃O₄ NPs could trigger the production of·OH efficiently in cells.We then explored the effect of Fe₃O₄ NPs on the cytotoxicity of H₂O₂ to cells by MTT assay.The result demonstrated that H₂O₂ showed obvious toxicity to cells and the viability was decreased to 74.3%after exposing to H₂O₂ at 100?M.In the presence of 2.3 nm Fe₃O₄ NPs,the cell viability was further decreased to42.1%.The enhanced toxicity should be attributed to the generation of·OH.Similar toxicity was observed in 4.2 nm Fe₃O₄ NPs group.3.To investigate the in vivo behavior of nanoparticles,we evaluated the toxicity of the nanoparticles through injection into ICR mice at a dose of 100 mg/kg,the results showed that only 2.3 nm and 4.2 nm Fe₃O₄ NPs groups mices died.The element concentration in different organs after NPs injection was measured by Electron-coupled plasma atomic emission spectrometry?ICP-AES?,and the results showed that the distribution was related to the particle size,2.3 nm Fe₃O₄ NPs have a large accumulation in the heart,and the 9.3 nm Fe₃O₄ NPs are mainly distributed in the liver.Then detect the in vivo ROS and·OH in the organs homogenization,the results showed the ROS concentrations in serum,heart,liver,spleen and lung increased significantly following administration of 2.3 nm Fe₃O₄ NPs for 2 h,which were 2.16,1.61,4.03,and 1.90 times as high as that of the control mice.And only 2.3 nm Fe₃O₄ could induce the production of·OH in vivo,especially the·OH concentration in heart increased by 2.56 times.It has been reported that the ultrasmall NPs were highly toxic to heart.With H&E stained images,the prominent cardiotoxicity and pulmonary toxicity can be observed as illustrared by the necrotic regions and acute inflammatory cells.The above results showed that the ultrasmall Fe₃O₄?<5 nm?was highly toxic,while no obvious toxicity was detected for the large-sized Fe₃O₄?>5 nm?,SiO₂ and Au NPs.Based on the results of in vivo and in vitro experiments,we speculate that the toxicity of ultrasmall Fe₃O₄?<5 nm?was caused by two steps:the ultrasmall induced ROS and the subsequent Fenton reaction.In addition,we also found that the nuclei targeting and distribution in vivo were related to the size of the nanoparticles.This study not only illustrates the potential toxicological mechanism of ultrasmall Fe₃O₄nanoparticles,but also presents a potential application of ultrasmall Fe₃O₄?<5 nm?in therapy.