A comparative study of neurotoxic potential of synthesized polysaccharide-coated and native ferritin-based magnetic nanoparticles

Aim

To analyze the neurotoxic potential of synthesized magnetite nanoparticles coated by dextran, hydroxyethyl starch, oxidized hydroxyethyl starch, and chitosan, and magnetic nanoparticles combined with ferritin as a native protein.

Methods

The size of nanoparticles was analyzed using photon correlation spectroscopy, their effects on the conductance of planar lipid membrane by planar lipid bilayer technique, membrane potential and acidification of synaptic vesicles by spectrofluorimetry, and glutamate uptake and ambient level of glutamate in isolated rat brain nerve terminals (synaptosomes) by radiolabeled assay.

Results

Uncoated synthesized magnetite nanoparticles and nanoparticles coated by different polysaccharides had no significant effect on synaptic vesicle acidification, the initial velocity of L-[¹⁴C]glutamate uptake, ambient level of L-[¹⁴C]glutamate and the potential of the plasma membrane of synaptosomes, and conductance of planar lipid membrane. Native ferritin-based magnetic nanoparticles had no effect on the membrane potential but significantly reduced L-[¹⁴C]glutamate transport in synaptosomes and acidification of synaptic vesicles.

Conclusions

Our study indicates that synthesized magnetite nanoparticles in contrast to ferritin have no effects on the functional state and glutamate transport of nerve terminals, and so ferritin cannot be used as a prototype, analogue, or model of polysaccharide-coated magnetic nanoparticle in toxicity risk assessment and manipulation of nerve terminals by external magnetic fields. Still, the ability of ferritin to change the functional state of nerve terminals in combination with its magnetic properties suggests its biotechnological potential.Superparamagnetic iron oxide nanoparticles are a promising candidate for increasing the efficiency of targeted drug delivery and therapy due to external magnetic guidance. Nanomaterials differ from those in bulk forms because they often show unexpected physical and chemical properties. They may produce potential functional and toxicity effects on human nerve cells due to their ability to pass through biological membranes and increase the risk of the development of neurodegenerative diseases (1-3). They can penetrate the blood-brain barrier (3-5) and kill nervous cells in vitro (6-8). Surface modification of iron oxide is a key issue for enhancing its interaction with the cell membrane. By using iron oxide nanoparticles coated by dextran, it was shown that labeled cells could be tracked by magnetic resonance imaging in vivo (9,10). Dextran occupies a special place among polysaccharides because of its wide application. Contrast agents based on dextran-coated iron oxides, eg, Endorem (Guerbet, Roissy, France) and Resovist (Bayer Schering Pharma AG, Berlin-Wedding, Germany), have been commercially available for human use as blood pool agents. Similarly, immortalized cells from the MHP36 hippocampal cell line labeled in vitro with gadolinium rhodamine dextran were tracked in ischemia-damaged rat hippocampus in perfused brains ex vivo (11).Taking into account that all nanoparticles are more or less toxic and the brain can be a target for their neurotoxic action (3,8,12,13), it is crucial to know their neurotoxic potential. Estimation of neurotoxic risks of nanoparticles can be assessed at various levels of nervous system organization. This research was conducted at the neurochemical level according to the Guidelines for Neurotoxicity Risk Assessment of US Environmental Protection Agency (14), assessing the uptake and release of the neurotransmitters in nerve terminals (15,16). It has been suggested that a possible target for nanoparticles, beyond the already established microglial cells, are presynaptic terminals of neurons (12). Presynaptic nerve terminals contain vesicular pool of neurotransmitters that can be released by exocytosis to the synaptic cleft in response to stimulation (17,18). A key excitatory neurotransmitter in the mammalian central nervous system is glutamate, which is implicated in many aspects of normal brain functioning. Abnormal glutamate homeostasis contributes to neuronal dysfunction and is involved in the pathogenesis of major neurological disorders (19,20). Under normal physiological conditions, extracellular glutamate between episodes of exocytotic release is kept at a low level, thereby preventing continual activation of glutamate receptors and protecting neurons from excitotoxic injury. Low extracellular glutamate concentration is maintained through its uptake by high-affinity Na⁺-dependent glutamate transporters located in the plasma membrane of neurons and glial cells.Prototypic nanoparticles have been shown to be useful for investigation of synaptic mechanisms underlying the development of neurotoxicity (8,12). Ferritin may be considered as a model nanoparticle (8,12) because it is composed of 24 subunits, which form a spherical shell with a large cavity where up to 4500 ions Fe³⁺ can be deposited as compact mineral crystallites resembling ferrihydrite (21-25). Ferritin stores cellular iron in a dynamic manner allowing the release of the metal on demand (24). Its cores exhibit superparamagnetic properties, which are inherent to magnetic nanoparticles, and vary in diameter from 3.5 nm to 7.5 nm in different tissues (26,27). This protein can penetrate blood-brain barrier (28) and be transported in different cells using clathrin-mediated endocytosis, similarly to many artificial nanoparticles that use the same mechanism (8,29,30).Recently, there has started an examination of ferritin from the biotechnological point of view. The hypothesis was that ferritin might be considered a good tool and prototypical nanoparticle for investigation of possible toxic properties of metal nanoparticles coated by dextran/polymer shells and possible causes of neurodegeneration associated with exposure to nanoparticles (8,12). Ferritin has been suggested as a label for high-gradient magnetic separation (31) and magnetic force microscopy imaging (32). Recently, it has been shown that the avascular microscopic breast and brain tumors could be noninvasively detected by designing nanoparticles that contained human ferritin as molecular probes for near-infrared fluorescence and magnetic resonance imaging (33).This research was focused on two aspects – the first was the assessment of neurotoxic potential of synthesized nanoparticles of magnetite (MNP) coated by dextran, hydroxyethyl starch, oxidized hydroxyethyl starch, chitosan as well as uncoated nanoparticles, studying their effects on: 1) the uptake of L-[¹⁴C]glutamate by rat brain nerve terminals via specific high-affinity Na⁺-dependent plasma membrane transporters; 2) the ambient level of L-[¹⁴C]glutamate in nerve terminals; 3) the membrane potential (Em) of the plasma membrane of nerve terminals using potential-sensitive fluorescent dye Rhodamine 6G; 4) transmembrane current across the planar lipid membrane using planar lipid bilayer technique; 5) acidification of synaptic vesicles in nerve terminals using pH-sensitive fluorescent dye acridine orange. The second aspect was a comparative analysis of neurotoxic potential of these synthesized polysaccharide-coated nanoparticles and ferritin, which could bring new insight into a possible usage of ferritin as an analogue of polymer-coated magnetic nanoparticle in toxicity risk assessment.