Toxicological assessment of silica-coated iron oxide nanoparticles in human astrocytes
Graphical abstract
Introduction
Iron oxide nanoparticles (ION) have great potential for an increasing number of medical and biological applications. Due to their size-driven superparamagnetism, they are used in diagnosis, therapeutics and tumor destruction (Revia and Zhang, 2016). ION versatility, together with their physicochemical properties, allow their use as magnetic resonance imaging agents (Abakumov et al., 2015; Gkagkanasiou et al., 2016), heating mediators in hyperthermia-based cancer therapy (Blanco-Andujar et al., 2016; Dan et al., 2015), and molecular cargo in targeted drug (Elzoghby et al., 2016; Thomsen et al., 2015) and gene delivery (Li et al., 2016). Particularly, the design of specific ION for diagnosis and treatment of neurodegenerative and neurovascular diseases has noticeably increased in the last years (Kanwar et al., 2012). However, studies on their possible neurotoxic effects are still scarce and inconclusive, especially in human models (Valdiglesias et al., 2016). The specific use of ION in diagnostics and therapies on the nervous system requires their introduction into the body; given their small size they can cross the blood brain barrier (BBB) and access the brain tissue (Win-Shwe and Fujimaki, 2011). This demonstrated ability of ION to cross the BBB, combined with their high surface area and reactivity, makes the nervous system extremely vulnerable to their potential toxicity. Indeed, previous studies have already demonstrated that ION may induce cytoskeleton impairment, plasmatic membrane disruption, oxidative stress, DNA damage, or alterations in cell signaling pathways in different cells form nervous origin (reviewed in Valdiglesias et al., 2016). Still, the lack of robust toxicological screenings, and poor comprehension of predictive paradigms of nanoneurotoxicity are the major obstacles in translating the advancing nanoparticle designs into viable biomedical platforms system (Kim et al., 2013).
Several uncoated and differently coated ION have been reported to be highly biocompatible nanomaterials which do not pose a serious threat to the organism (Laurent et al., 2014). However, despite being considered as generally safe, potential ION toxicity cannot be completely discarded since results from studies on this regard are often contradictory, and ION effects at certain levels, such as genotoxicity or carcinogenicity, have been poorly addressed (reviewed in Valdiglesias et al., 2014; Revia and Zhang, 2016). Most studies reported so far on the consequences of in vitro exposure of nervous system cells to different uncoated and coated ION have been performed in neurons, particularly PC12 rat and SH-SY5Y human cells (Deng et al., 2014; Imam et al., 2015; Kiliç et al., 2015; Wu et al., 2013; Wu and Sun, 2011). However, investigating the potential harmful effects of ION on other different nervous system cells (i.e. glial cells: astrocytes, oligodendrocytes and microglial cells) is also relevant since they are involved in neuron support and protection, and alterations in these cells have been implicated in the onset and progression of several neurodegenerative diseases (Barker and Cicchetti, 2014; Cai and Xiao, 2016; Phatnani and Maniatis, 2015). Astrocytes are especially interesting since they are the most abundant brain cell type and the first cellular obstacle ION interact with, and are strategically distributed between the blood vessels and neurons (Geppert et al., 2011). Besides, they seem to play a key role in the etiology of neurodegenerative disorders and, consequently, have been proposed as new targets for the treatment of important neuropathologies such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease (Finsterwald et al., 2015).
For in vivo purposes, nanoparticles are required to be biocompatible, water-dispersible, stable in biological media, and uniform in size to maintain the suitable magnetic properties (Chang et al., 2007; Lee et al., 2015). Surface coatings are known generally to influence advantageously nanoparticle features. For this reason, ION are often coated with different organic and inorganic materials to increase their stability and improve their biocompatibility and biodegradability (Al Faraj et al., 2015), decrease their cytotoxicity (Magdolenova et al., 2013), and provide an ample surface for functionalization (Revia and Zhang, 2016). Among all the possible coating materials, silica has several advantages that makes it very suitable for biomedical applications. It can increase ION biocompatibility without affecting magnetic properties, may convert hydrophobic nanoparticles into hydrophilic water-soluble particles, helps to prevent aggregation by improving the nanoparticle chemical stability, and the silanol-terminated surface groups may be modified with various coupling agents to covalently bind to specific ligands reviewed in Andrade et al. (2009). All these properties make silica one of the most commonly used agents for ION coating, particularly for bioimaging and biosensing purposes (Alwi et al., 2012). Still, the possible neurotoxicity of silica-coated ION (S-ION), particularly on nervous cells different from neurons, has not been discarded yet.
ION toxicity has been demonstrated to vary considerably and also to depend on cell type and physical-chemical characteristics such as size, shape, presence/type of coating, and stability in biological media (Gupta and Gupta, 2005; Mahdavi et al., 2013; Strehl et al., 2016). In a previous study conducted by our research group (Costa et al., 2015), effects induced by S-ION on viability of A172 glial cells and SH-SY5Y neuronal cells were evaluated. Results showed that S-ION significantly decreased viability, with a moderate effect in the glial cells; besides, a serum-protective effect was observed for both cell lines. Moving forward, the main objective of the present work was to investigate for the very first time the effects of S-ION on human astrocytes (A172 glioblastoma cells), in order to obtain an overview of the risk these nanoparticles may pose when used in biomedical applications on the human nervous system. To this aim, a complete toxicological screening was performed to assess S-ION effects at different levels, including cytotoxicity – cellular membrane impairment, cell cycle disruption and cell death production – and genotoxicity – H2AX phosphorylation, primary DNA damage and micronuclei (MN) induction –, considering also alterations in DNA repair ability and iron ion release capacity.
Section snippets
Chemicals
Bleomycin (BLM) (CAS no. 9041-93-4) and Triton X-100 (CAS no. 9002-93-1) was purchased from Panreac AppliChem, and mytomycin C (MMC) (CAS no. 50-07-7), camptothecin (Campt) (CAS no. 7689-03-4), hydrogen peroxide (H2O2) (CAS no. 7722-84-1), and propidium iodide (PI) (CAS no. 25535-16-4) were purchased from Sigma-Aldrich Co.). BLM, MMC, and PI were dissolved in sterile distilled water, and Campt was dissolved in DMSO prior to use.
Nanoparticle preparation and characterization
S-ION were synthesized and prepared as stable water suspensions
Results
A complete physical-chemical characterization of these nanoparticles was previously carried out (Costa et al., 2015). Briefly, S-ION used are spherical particles with an average diameter of 20.2 nm, including core and silica coating; less than 2% of the S-ION surface presents iron, confirming an effective silica coating. Mean hydrodynamic size and zeta potential values in different media demonstrated the suspension stability and low tendency to agglomeration.
Discussion
To guarantee their safety, nanoparticles must not be toxic to the cells at concentrations suitable for magnetic targeting or other biomedical applications. Previous studies indicated that ION exhibit very subtle or no cytotoxic activity when administered at concentrations remaining below 100 μg/ml (Laurent et al., 2014). However, it has also been demonstrated in a number of in vitro and in vivo studies that ION, both naked and differently coated, may induce adverse effects, even at low doses,
Conclusions
Despite the increasing use of ION in biomedical applications, many current studies on toxicity assessment are far from reaching a conclusion and providing guidance for their safe use. Hence, more comprehensive methodological approaches need to be addressed for the evaluation of ION, in order to better understand the potential risk they may pose. In the present study genotoxicity and cytotoxicity associated with S-ION exposure were evaluated in glial cells by a battery of assays. Results
Conflicts of interest
The authors declare that they have no conflict of interests.
Acknowledgements
This work was funded by Xunta de Galicia (ED431B 2016/013). V. Valdiglesias was supported by a Xunta de Galicia postdoctoral fellowship (reference ED481B 2016/190-0). N. Fernández-Bertólez was supported by an INDITEX-UDC fellowship. F. Brandão is supported by the grant SFRH/BD/101060/2014, funded by FCT (financing subsided by national fund of MCTES). Authors would also like to acknowledge COST Action CA15132 “The comet assay as a human biomonitoring tool (hCOMET)”.
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These authors contributed equally to the senior authorship of this manuscript.