A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron oxide nanoparticles

https://doi.org/10.1016/j.colsurfb.2009.08.044Get rights and content

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) are increasingly used in medical applications, such as targeting delivery and imaging. In the future, patients are more likely to be exposed to pharmaceutical products containing such particles. The study of toxicity of SPIONs has become of great importance in recent years, although the published data in this arena is limited. The aim of the present work is to investigate the cytotoxicity of SPIONs and the effect of the particles on the cell medium components. For this purpose, uncoated and polyvinyl alcohol (PVA) coated SPIONs with narrow size distribution were synthesized via a well-known coprecipitation method. The mouse fibroblast cell line L929 was exposed to SPIONs to probe the toxicity of magnetic nanoparticles during the bio application. Changes to the cell medium caused by SPIONs were analyzed with zeta potential measurements, ultraviolet visible spectroscopy (UV/vis) and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. It is observed that gas vesicles are formed in SPION-treated cells. Toxicity is conventionally explained by changes in the DMEM's pH and composition due to the tendency of SPIONs to interact with biomolecules. A new procedure is proposed to examine the in vitro toxicity of nanoparticles in a more rigorous manner, which gives an improvement in the relationship between in vivo and in vitro toxicity studies.

Introduction

The efficiency of cancer chemotherapy depends not only on the anticancer agent used, but also on the manner that it is delivered to target tissues and cancer cells [1], [2]. Nanotechnology has offered new alternatives for cancer diagnosis and targeted treatments because of the unique properties of nanoscale structures. Such nanoscale structures include gold, iron oxides (i.e., magnetite and maghemite) and titanium oxide. Recent reports demonstrate that drug-coated polymeric nanospheres and nanocapsules can increase intracellular anticancer effects [8], [9], [10], [11]. Furthermore, new nanocomposites are used for DNA detection [3], intracellular labeling [4], drug delivery [5], cancer targeting [6] and imaging [7].

For in vivo applications, it is of utmost importance that nanoscale structures are biocompatible. In nanoparticles, for example, it is important that the coating is non-toxic and insulates the body from undesired toxic side effects. Specifically, researchers have studied undesired toxic side effects, often with a focus on metals and metal oxide nanoparticles [11], [12], [13], [14], [15]. Jeng and Swanson [11] have reported changes in cellular morphology, mitochondrial function, membrane leakage (by lactate dehydrogenase (LDH)), permeability of plasma membrane and apoptosis after exposing cells to several types of metal oxide nanoparticles. Zinc oxide particles were found to be the most potent. Hussain et al. [13] showed toxic effects on cellular morphology, mitochondrial function (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) after exposure of rat liver cells to Ag nanoparticles. Fe3O4 and TiO2 nanoparticles, however, were shown to be much less toxic [13]. Veranth and co-workers [15] studied the cytokine response in BEAS-2B cells due to metal oxide nanoparticles and found that metal oxide particles have a lower toxicity than an equal mass of micron-sized particles of the same nominal composition.

The successful drug delivery and transfections can have massive academic, clinical, and practical impacts on gene therapy, cell and molecular biology, pharmaceutical and food industries, and bio-production [16], [17], [18]. Targeting specific sites in vivo for the delivery of therapeutic agents presents a major obstacle to the treatment of many diseases, including cancer. A targeted delivery technique that has gained prominence in recent years is the use of magnetic nanoparticles. In these systems, therapeutic agents are attached to biocompatible magnetic nanoparticles and magnetic fields generated outside the body are focused on specific targets in vivo [19], [20]. Superparamagnetic iron oxide nanoparticles (SPIONs) are promising candidates in a number of biomedical applications, including enhanced resolution magnetic resonance imaging (MRI), hyperthermia, drug delivery, tissue repair, cell and tissue targeting and transfection [21], [22]. The potential of SPION in these applications is due to their small size (below 30 nm), their superparamagnetic properties, and their generally good biocompatibility. Not all researchers, however, seem to agree on the non-toxicity of SPIONs, and different assays have been used to investigate these concerns. The MTT assay has been used to measure the toxicity of SPIONs [19], [20], [21]. One of the toxic effects induced by nanoparticles is their ability to cause oxidative stress [23], [24]. There is a link between DNA damage, mutations and cancer, and nanoparticles that are potent in causing this damage are more likely to have an adverse effect in cancer development. Reported studies on the effect of nanoparticles on DNA damage and oxidative stress are scarce [25].

In general, it is preferable to investigate the toxicity of nanoparticles with in vitro assays because they are simpler, faster, more cost effective and pose no ethical problems compared to in vivo studies. However, researchers found little correlation between in vivo and in vitro toxicity results, especially with using nanoparticles [26]. Toxic responses were observed for nanoparticles in vitro [27] but the same results were not exactly reproduced in vivo [28]. One possible reason for this discrepancy may be that during in vivo assays, there will be few detectable changes observed due to the well-known principle of body homeostasis. However; there is no such phenomenon in vitro. This toxicological finding is not so far from its pharmacologic analogy where in vitro results may not support in vivo results. The poor correlation between the toxicity measurement methods for in vivo and in vitro may also be due to the ability of nanoparticles to change the cell medium during in vitro assays, for example by protein/ion adsorption and pH changes, caused in part by the SPIONs surface activity. In contrast, during in vivo assays, changes in the surrounding tissues do not occur to the same extent when identical amounts of nanoparticles are applied. As a more specific example, the kidneys are able to filter blood and produce urine, thereby regulating the water and ion concentrations of blood plasma. Besides excreting nitrogen compounds, toxins, water, and electrolytes, kidneys also act as endocrine organs by secreting the hormones erythropoietin, renin and prostaglandins [29]. In order to obtain comparable results between the in vivo and in vitro cytotoxicity of SPIONs, the effects of SPIONs on the cell culture medium should be probed.

The aim of this study is to advance the study of the cytotoxic effects of magnetite nanoparticles and to probe their ability to change cell culture medium compositions. To this end, the mouse connective tissue cells L929 were used and toxicity levels and changes in the cell culture medium compositions due to SPIONs exposure were determined using the MTT assay and UV/vis spectroscopy. A new procedure is proposed to examine the in vitro toxicity of nanoparticles that can lead to more reliable toxicity results.

Section snippets

Particle preparation and characterization

Polyvinyl alcohol (PVA, MW = 30,000–40,000, degree of hydrolysis 86–89%) and the dye crystal violet were purchased from Fluka (Switzerland). Analytical grade ferrous and ferric chloride (FeCl2 and FeCl3) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany) and used without further purification. Other chemicals were of analytical grade.

Solutions were prepared by bubbling argon through deionized (DI) water for 30 min for deaeration. Iron salts were dissolved in DI water

Characteristics of the prepared SPIONs

Fig. 1 shows TEM images of uncoated and coated SPION. For the coated nanoparticles, magnetic beads with diameters between 20 and 30 nm are formed, while single aggregated nanoparticles are observed for the uncoated iron oxide nanoparticles. The composition of magnetite is confirmed via selected area electron diffraction (SAED) (Fig. 1(c)). Fig. 2 shows XRD patterns of the uncoated and coated nanoparticles. The full width at half maximum (FWHM) of the (3 1 1) reflection was used to determine the

Conclusion

The core hypothesis of this study was to make a direct correlation between in vitro and in vivo studies by understanding the effect of nanoparticles on the cell medium, in particular the interaction of nanoparticles with biomolecules. The effects of both PVA coated and uncoated superparamagnetic iron oxide nanoparticles on the cell medium were examined. It is shown that the conventional in vitro examination method may contain large errors as compared to the modified method. This may be

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