Background: Iron oxide nanoparticles have been approved by food and drug administration for clinical application as magnetic resonance imaging (MRI) and are considered to be a biocompatible material. Large iron oxide nanoparticles are usually used as transversal (T₂) contrast agents to exhibit dark contrast in MRI. In contrast, ultrasmall iron oxide nanoparticles (USPIONs) (several nanometers) showed remarkable advantage in longitudinal (T₁)-weighted MRI due to the brighten effect. The study of the toxicity mainly focuses on particles with size of tens to hundreds of nanometers, while little is known about the toxicity of USPIONs. Results: We fabricated Fe₃O₄ nanoparticles with diameters of 2.3, 4.2, and 9.3 nm and evaluated their toxicity in mice by intravenous injection. The results indicate that ultrasmall iron oxide nanoparticles with small size (2.3 and 4.2 nm) were highly toxic and were lethal at a dosage of 100 mg/kg. In contrast, no obvious toxicity was observed for iron oxide nanoparticles with size of 9.3 nm. The toxicity of small nanoparticles (2.3 and 4.2 nm) could be reduced when the total dose was split into 4 doses with each interval for 5 min. To study the toxicology, we synthesized different-sized SiO₂ and gold nanoparticles. No significant toxicity was observed for ultrasmall SiO₂ and gold nanoparticles in the mice. Hence, the toxicity of the ultrasmall Fe₃O₄ nanoparticles should be attributed to both the iron element and size. In the in vitro experiments, all the ultrasmall nanoparticles (< 5 nm) of Fe₃O₄, SiO₂, and gold induced the generation of the reactive oxygen species (ROS) efficiently, while no obvious ROS was observed in larger nanoparticles groups. However, the ·OH was only detected in Fe₃O₄ group instead of SiO₂ and gold groups. After intravenous injection, significantly elevated ·OH level was observed in heart, serum, and multiple organs. Among these organs, heart showed highest ·OH level due to the high distribution of ultrasmall Fe₃O₄ nanoparticles, leading to the acute cardiac failure and death. Conclusion: Ultrasmall Fe₃O₄ nanoparticles (2.3 and 4.2 nm) showed high toxicity in vivo due to the distinctive capability in inducing the generation of ·OH in multiple organs, especially in heart. The toxicity was related to both the iron element and size. These findings provide novel insight into the toxicology of ultrasmall Fe₃O₄ nanoparticles, and also highlight the need of comprehensive evaluation for their clinic application. Graphical

With the potential uses of photothermal therapy (PTT) in cancer treatment with excellent efficacy, and the growing concerns about the nanotoxicity of hyperthermia agents such as carbon nanotubes and gold-based nanomaterials, the importance of searching for a biocompatible hyperthermia agent cannot be emphasized too much. In this work, a novel promising hyperthermia agent employing magnetic Fe₃O₄ particles with fairly low toxicity was proposed. This hyperthermia agent showed rapid heat generation under NIR irradiation. After modification with carboxymethyl chitosan (CMCTS), the obtained Fe₃O₄@CMCTS particles could disperse stably in PBS and serum without any aggregation. The modification of CMCTS could decrease the adsorption of bovine serum albumin (BSA) and improve the cellular uptake. In a comparative study with hollow gold nanospheres (HAuNS), Fe₃O₄@CMCTS particles exhibited a comparable photothermal effect and fairly low cytotoxicity. The in vivo magnetic resonance (MR) images of mice revealed that by attaching a magnet to the tumor, Fe₃O₄@CMCTS particles accumulated in the tumor after intravenous injection and showed a low distribution in the liver. After being exposed to a 808 nm laser for 5 min at a low power density of 1.5 W cm⁻², the tumors on Fe₃O₄@CMCTS-injected mice reached a temperature of ∼52 °C and were completely destroyed. Thus, a kind of multifunctional magnetic nanoparticle with extremely low toxicity and a simple structure for simultaneous MR imaging, targeted drug delivery and photothermal therapy can be easily fabricated.