Elsevier

Biomaterials

Volume 32, Issue 35, December 2011, Pages 9401-9414
Biomaterials

The effect of static magnetic fields on the aggregation and cytotoxicity of magnetic nanoparticles

https://doi.org/10.1016/j.biomaterials.2011.08.075Get rights and content

Abstract

Biomedical applications of magnetic nanoparticles (MNP), including superparamagnetic nanoparticles, have expanded dramatically in recent years. Systematic and standardized cytotoxicity assessment to ensure the biosafety and biocompatibility of those applications is compulsory. We investigated whether exposure to static magnetic field (SMF) from e.g. magnetic resonance imaging (MRI) could affect the cytotoxicity of superparamagnetic iron oxide (SPIO) nanoparticles using mouse hepatocytes and ferucarbotran, a liver-selective MRI contrast agent as a model system. We show that while the SPIO satisfied the conventional cytotoxicity assessment, clinical doses combined with SMF exposure exerts synergistic adverse effects such as reduced cell viability, apoptosis, and cell cycle aberrations on hepatocytes in vitro and in vivo. Concomitant treatments with the SPIO and SMF generated SPIO aggregates, which demonstrated enhanced cellular uptake, was sufficient to induce the cytotoxicity without further SMF, emphasizing that the SPIO aggregates were the predominant source of the cytotoxicity. Interestingly, the apoptotic effect was dependent on levels of reactive oxygen species (ROS) and SPIO uptake while the reduced cell viability was independent of these factors. Moreover, long-term monitoring showed a significant increase in multinuclear giant cells in the cells concomitantly treated with the SPIO and SMF compared with the control. The results demonstrate that the SPIO produces unidentified cytotoxicity on liver in the presence of SMF and the SPIO aggregates predominantly exert the effect. Since aggregation of MNP in biological milieu in the presence of strong SMF is inevitable, a fundamentally different approach to surface fabrication is essential to increase the biocompatibility of MNP.

Introduction

The diverse biomedical applications of magnetic nanoparticles (MNP) have expanded dramatically in recent years. These applications include magnetic resonance contrast agents (MR CA), tracking of implanted cells, drug delivery, hyperthermia, biosensors, and bioseparation [1], [2]. Superparamagnetic nanoparticles (SPNP) have been successfully used as MR CA, and this has been their principal clinical use. Commercial superparamagnetic iron oxide nanoparticles (SPION) such as ferucarbotran and ferumoxide, which potentially yield higher contrast enhancement than conventional Gd-based CA, had been competitive with conventional agents for a decade and are still used commonly in recent studies [3], [4]. These SPION agents are liver-specific due to uptake by Kupffer cells’ in the reticuloendothelial system of the liver.

The various nanomaterials being engineered as part of the novel and rapidly evolving field of nanotechnology are expected to make enormous positive contributions to human society. However, this unprecedented technology also creates profound biosafety concerns due to the unique physicochemical properties of nanomaterials, such as their small sizes, shapes, unidentified surface reactions and uncontrollable aggregation [5], [6]. Direct biomedical applications of nanomaterials such as SPNP may be outstanding examples of the positive and negative aspects of nanotechnology, as they have recently been shown to be associated with the cellular toxicity [2], [7]. In this regard, non-toxicity is probably the most essential requirement for SPNP used for biomedical applications. To this end, synthesized SPNP are routinely coated before use with appropriate moieties or ligands to reduce cellular toxicity and increase biocompatibility [1], [8]. Such modifications are in fact quite effective for minimizing cellular toxicity. A representative species of SPION that has been used for MR CA and tracking of stem cells, ferucarbotran, is coated with carboxydextran. This modification is known to reduce cellular toxicity and aggregation in vitro and in vivo sufficiently for clinical use [3], [4]. Although several adverse side effects of ferucarbotran are recognized, including allergic reaction (diffuse erythematous rash), and reductions of neutrophils and Factor XI, these have been considered marginal and tolerable in patients [9]. However, one very recent study showed that in mice, a single injection of ferucarbotran at clinically relevant dose induced a remarkable degree of apoptosis and subsequent depletion of the Kupffer cells in the liver, suggesting the possibility of further, as yet unidentified health problems [10]. Given the lack of specialization of liver cells and the wide variety of metabolic processes performed by hepatocytes, it is necessary to investigate whether SPIO also has unsuspected adverse effects on hepatocytes.

Electromagnetic biology is a field that explores the effects of electric and/or magnetic fields on living organisms. Decades of study in this field have convincingly demonstrated that diverse species of birds, sea turtles, and butterflies navigate for long distances, and mammals align on the ground, by sensing the direction of the geomagnetic field [11], [12]. Apart from the effects of the geomagnetic field on animal behavior, electromagnetic biology also investigates potential adverse effects of various environmental electromagnetic fields (EMF), a matter of growing public concern. The representative EMF are those from power lines and mobile phones, as well as static magnetic fields (SMF) from magnetic resonance imaging systems and magnetic levitation [13]. In particular, accumulating evidences has shown that SMF treatments induce apoptosis, genotoxicity, and cell differentiation in various kinds of cells [13], [14]. However, no previous study has been properly conducted to investigate the effect of MNP combined with SMF exposure on cellular toxicity in vitro and in vivo. From the perspective of electromagnetic biology, it is reasonable to examine the cytotoxicity of MNP in combination with SMF exposure, because MNP such as SPNP are magnetized in the presence of SMF and might possibly exert unidentified adverse effects on living organisms.

In this study, we investigated whether exposure to strong SMF from e.g. MRI could affect the cellular toxicity of SPION in vitro and in vivo using mouse hepatocytes as a model cell type.

Section snippets

Cell culture, reagents, static magnetic field, and SPIO aggregates

A normal mouse liver cell line, NCTC 1469, purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured at 37 °C under a humidified atmosphere containing 5% CO2 in Dulbecco’s MEM (DMEM) supplemented with 10% horse serum (GIBCO BRL, Gaithersburg, MD, USA), 1% sodium pyruvate, and 100 U/ml penicillin/streptomycin, as previously described [15], [16]. Reagents not otherwise specified were obtained from Sigma (St Louis, MO, USA). The SPIO ferucarbotran (SHU 555A, Resovist™)

Cytotoxicity of SMF and SPIO on hepatocytes

Cultured normal mouse hepatocytes were treated with ferucarbotran and/or SMF delivered by permanent magnets in order to determine whether the cellular toxicity of SPIO is affected by SMF exposure. Cell viability was decreased in proportion to the concentration and intensity of ferucarbotran and SMF exposure, respectively, and also with the duration of treatments (Fig. 1A−D and SI Fig. 1A and C). Interestingly, concomitant treatment with the SPIO (0.5 mM) and SMF exposure (0.4 T) showed an acute

Electromagnetic biology perspective on nanotoxicity assessment of MNP

Recent increases in professional and public concern about the possible adverse effects of nanomaterials and nanotechnology have necessitated intense and systematic scientific examination of these issues. However, very limited attention has been paid to the potential negative effects of strong SMF and radiofrequency electromagnetic field (RF EMF) exposure from MRI scans in conjunction with SPION. A very recent report pointed out the paucity of conventional assessment of iron oxide NP toxicity as

Conclusion

This study provides evidence that ferucarbotran which is commonly used in various current studies and had been applied worldwide as liver-specific MR CA, has potential for iatrogenic cytotoxicity. Concomitant treatment with SPIO and SMF exposure produced SPIO aggregates that induced previously unsuspected cytotoxic effects both in vitro and in vivo. Even at sub-clinical concentrations of SPIO, the SPIO and SMF exposure treatments showed synergistic cytotoxic effects including reduced cell

Acknowledgements

This research was supported by Basic Science Research Program (Grant No. 2009-0076998) and Nuclear R&D Program (Grant No. 2009-0081817) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST). This work was also partly funded by The Advanced Medical Technology Cluster for Diagnosis & Prediction, KNU from MKE, ROK (Grant No. RTI04-01-01).

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