Quercetin Attenuates Neurotoxicity Induced By Iron Oxide Nanoparticles Part 2

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Iron in dementia-associated diseases Iron and Alzheimer's disease

AD is a progressive brain disorder that slowly destroys learning, memory, and thinking skills. Age, gender, genetic susceptibility, lifestyle, and several pathological conditions such as diabetes and stroke as well as brain iron accumulation are risk factors related to AD [87, 88]. Senile plaques contain aggregates of extracellular amyloid-beta (A) oligomers and neurofibrillary tangles(NFTs)contain aggregates of intracellular abnormal hyperphosphorylated tau protein are two common pathological hallmarks of AD. There is a relationship between iron accumulation and pathological hallmarks of AD. Abnormal levels of iron in the hippocampus and cortex of AD-affected subjects have been reported 75]. An in vivo study indicates iron deposits accompanied by senile plaques in the brain of a transgenic mouse model of AD by quantitative susceptibility mapping(QSM), a new technique in MRI [89]. The early plaques were formed in parallel with iron overload in a mouse model of AD[90]. Fe+ within senile plaques 

can be converted to a more reactive form of iron, Fe2+, by Aβ [78]. On the other hand,4-HNE raised from lipid peroxidation directly reacts with Aβ and produces oxidation products, which leads to Aβ aggregation [76]. Also, the Aβ peptide directly produces H, O, in an iron reduction-dependent process, a process that exacerbates oxidative stress and iron overload [91]. Iron can increase the expression of amyloid precursor protein (APP)by affecting the IRE site of APP mRNA.Furthermore, iron can bind to Aβ and increase Aβ aggregation 92]. The relationship between iron deposition and tau phosphorylation has been demonstrated via cortical imaging by QSM and tau Positron Emission Tomography scanning (tau-PET)in AD subjects [93]. Iron promotes the phosphorylation of tau by activating the cyclin-dependent kinase(CDK5)/P25 complex and glycogen synthase kinase-3β(GSK-3β)to form NFTs and decrease the efflux of iron ions[92]. According to these explanations, it can be concluded that there is a positive feedback loop among iron accumulation, oxidative stress, Aβ aggregation, and tau hyperphosphorylation. Researchers could reduce the toxicity of the plaques, enhance the solubility of Aβ, and reduce the formation of NFTs by eliminating the iron ions by using iron chelators.

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Iron and Parkinson's disease

PD is another neurodegenerative disease characterized by motor symptoms. Cognitive decline usually happens two decades before the diagnosis of motor symptoms. Hence, early diagnosis with considering cognitive decline can partly prevent the progression of PD[94]. PD occurs due to the degeneration of dopamine neurons particularly in a part of the substantia nigra called the pars compacta. Considerably, the loss of dopamine 

in the pars compacta disrupts voluntary motor control, increases the overall excitatory drive in the basal ganglia, and causes the characteristic symptoms of PD. Within the synapse, dopamine can be broken down and inactivated by two enzymes including monoamine oxidase(MAO)and catechol-O-methyl transferase (COMT)[95]. MAO activity is known to affect iron lev-els in animals and humans. There are complex interactions between free iron levels and MAO in the brain. However, increased oxidative stress appears to be a link between MAO, iron level, and neuronal damage. H, O2 is a normal product of monoamine oxidation via MAO. H, O, can participate in the Fenton reaction and produce highly active free radicals. In aging, MAO and brain iron levels increase which leads to an increase in components of the Fenton reaction and damage of macromolecules [96]. Thus inhibition of MAO or removal of the Fe²ions by an iron chelator is two approaches with the same goal in PD patients at the same time, increasing the monoamine levels, decreasing components of the Fenton reaction, and the consequent oxidative stress.

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Like AD, hyperphosphorylated tau and a decrease in soluble tau can cause iron overload in neurons via a decrease in APP-mediated iron export, which may be one of the causes of memory dysfunction in PD[97]. Besides, iron deposition was observed in structures supporting cognitive functions such as the hippocampus [85]. Evidence collected from 1988 to 2008 by A Jon Stoessl et al. showed abnormal deposition of iron, which is mainly together with ferritin in the substantia nigra neurons, motor-related area of PD patients. This data showed iron concentration is directly related to the severity of the disease [98]. Lewy bodies and Lewy neurites composed of abnormal a-Synuclein filaments are the most important neuropathological characteristics of PD [94]. At the molecular level, there is a close relationship between a-Synuclein aggregation and iron accumulation. Fe3+from the Fenton reaction directly induces a-synuclein expression and aggregation. Overexpression of hepcidin, a potential regulator of iron transporters, reduces the accumulation of iron in the brain and Fenton reaction thereby a-Synuclein aggregation and ROS production are reduced in the high-risk areas of the brain related to dementia and motor disorders [99, 100]. Thus the application of iron chelators that enhances the expression of hepcidin may inhibit a-synuclein aggregation.

Iron and stroke

There is evidence for crosstalk between certain types of stroke, iron overload, and memory dysfunction [86, 101, 102]. Stroke is one of the major causes of memory dysfunction, and nearly 30% of stroke patients develop dementia within 1 year of stroke onset [103]. Athero-sclerosis, diabetes, hypertension, smoking, high BMI, and dyslipidemia are risk factors for ischemic stroke [104]. Several mechanisms are involved in brain injuries induced by ischemia including inflammation, oxidative stress, the elevated concentration of intracellular calcium, enhanced excitatory amino acids, and increased levels of free iron and ferritin [105]. Post-stroke memory dysfunction can also be caused by vascular dementia, AD pathology [103], iron overload, and oxidative stress [86]. Edema formation by excess iron induces oxidative cell damage after a hemorrhagic stroke [106]. Iron deposition accompanied by a decrease in GSH and GPX and an increase in lipid peroxidation have been reported in neurons of ischemic stroke models [83]. Kondo et al.reported iron deposition in the hippocampus, striatum, and cerebral cortex in rats with transient forebrain ischemia. Late and early lipid peroxidation due to iron deposition after ischemia might be one of the causes of neuronal cell death [107]. Low oxygen condition caused by ischemic stroke leads to more iron influx into the brain. On other hand, acidic pH caused by ischemic stroke leads to dissociation of Fe3 from transferrin and its reduction to Fe2+, thereby NTBI uptake occurs. Neurons uptake NTBI and undergo Fenton/Haber-Weiss reaction, which produces harmful reactive radicals species and leads to lipid peroxidation and neuronal cell death [55].

IONPs metabolism-induced neurotoxicity

IONPs consist of an iron oxide core and a protective coating [108, 109]. Iron oxides have several chemical structures such as magnetite (Fe, O)), maghemite y-Fe, O3), hematite (a-Fe,O:), and wustite(FeO)[108]. Among them, Fe, Land Y-Fe, O, are more widely used in nanomedicine [14]. Despite the great similarities between these two iron oxides, Fe, O, is more magnetic and less stable than y-Fe, O,[110].Bare IONPs accumulated upon entering the circulation due to hydrophobic interactions between themselves. IONPs accumulation stimulates the immune system thereby IONPs can be destroyed in an opsonization-dependent mechanism.

Thus, a protective coating seems necessary for optimizing properties of IONPs including stability, biocompatibility, multi-functionalization, optimal biodegradation, hydrophilic interactions, and solubility [109]. Two types of IONPs are usually used for nanomedicine: superparamagnetic iron oxide nanoparticles (SPIONs) with a diameter of 50-100 nm and ultra-small superparamagnetic iron-oxide nanoparticles(USPIONs)with a diameter of up to 50 nm [lll]. IONPs can enter the human body by many administration routes including intravenous (IV), intramuscular (IM), subcutaneous, intrathecal, intra-tumoral, oral, and nasal. Several mechanisms are proposed for IONPs uptake by cells such as passive diffusion, phagocytosis, and types of endocytosis whether dependent or independent from clathrin and caveolae [112]. The entrance route of IONPs into the cell depends on their physicochemical properties such as size, shape, type of coating, and functional group of these particles[113-115]. IONPs have a nanoscale size and high surface-to-mass ratio. Despite being an advantage, these properties can cause more reactivity and cytotoxicity [116]. Several studies have been performed on the possibility of IONPs toxicity in various tissues, especially neural cells. Despite being improving memory disorders, their relative role in neurodegeneration and exacerbating memory disorders have been somewhat discussed. Cytotoxicity of IONPs depends on physicochemical properties including size, shape, type of coating, surface charge, exposure time/concentration, functional groups, and also type of cell treated with IONPs [14, 117]. Besides, it has been reported that the oxidation state of Fe ions in the iron oxide core determines the cytotoxicity of IONPs. Fe; O4 due to high potential oxidation has shown more genotoxicity than y-Fe, O, in the A549 human lung epithelial cell [112]. Although, evidence from several studies suggests that IONPs containing Fe, O2core had lower toxicity in comparison with y-Fe, O, due to their quick clearance from the body [14,118].In general, the major source of IONPs toxicity is the iron ions released from the core [119]. These iron ions along with other by-products of IONPs metabolism can interfere with iron homeostasis. In vivo studies indicated that liver ferritin levels enhanced after IONPs treatment, suggesting that IONPs are degraded, and their metabolic products induced alterations in iron responses [120, 121]. IONPs pass through the BBB by internalization mechanisms or destruction of endothelial cell membranes [14]. Iron uptake resulting from NPS metabolism depends on the levels of TfR expression on the cell surface [122]. IONPs have been reported to cross the BBB by interacting with the TfR on the abluminal membrane of endothelial cells. Also, BBB disruption and ROS enhancement caused by exposure to 10 μg/ml of Fe-NPs(10 and 30 nm) for 24 h in artificial BBBs have been reported [121]. In this regard, Jain et al. reported that IV administration of MNP（10 mg of Fe/kg in 100 μL of saline) in earlier time points did not change the levels of iron in the rat brain. Over time, binding of the released iron-transferrin complex to TfR on the BBB leads to an increase in iron content of the brain, especially one week after the MNP injection [122]. Thus, the level of TfR expression on the cell is another factor that differentiates NP uptake. Following the internalization of IONPs within the cell, they are placed in the acidic environment of the lysosome and metabolized resulting in the release of free iron ions into the cytosol. This degradation begins from the surface of NPs and gradually continues to their core. Released iron ions can participate in Fenton/Haber-Weiss reactions. The consequences of this event are manifested by the generation of early and secondary oxidation products that could damage cellular components such as nucleic acids, proteins, lipids, mitochondria[112, 123], and finally cause apoptosis[14,124]. Thus, it is proven that CNS can be affected by IONPs. These conditions are somehow related to neurodegeneration [121]. During neurodegenerative diseases in which the BBB becomes permeable to many elements, especially NPS, the use of IONPs can exacerbate the disease [14]. There is evidence of NPs toxicity in dementia-associated diseases such as AD, PD[121], and stroke|125|.In vitro model of AD indicates iron oxide-based NPs can aggravate the condition by forming complexes with Aβ [126]. The c-Abl tyrosine kinase plays a key role in neuronal cell death in PD. The c-Abl activation, increased α-synuclein, reduced cellular proliferation, increased ROS, and mitochondrial permeability has been reported in neurons after SPIONs treatment by Imam et al. [121].Leakage of electrons to the cytosol due to mitochondrial permeability causes a substantial reduction of striatal dopaminergic neurons in rats [121].Iron depositions induced by IV injection of USPIONs [2 mmol iron/kg body weight(0.15 ml)] have been observed in the stroke mouse model. It has also been shown that USPIONs can access the brain parenchyma and CSF by crossing the BBB, which was found via detection of USPIONs in meningeal macrophages and phagocytes in CSF-bathed areas [125].

Iron concentration in the brain is not static and is affected by factors such as age, a poor iron diet, iron deficiency anemia, and iron overload disorders. The iron content of different regions of the brain varies. Macro divisionally the white matter has a higher concentration of iron. Local divisionally, globus pallidus, red nucleus, substantia nigra, caudate-putamen, and dentate nucleus have a higher concentration of iron [l27]. Several studies have examined the tissue distribution of IONPs in the brain. Also, there is evidence for toxicity induced by coated IONPs. Frequent IV administration of ferumoxytol(8 mg/kg)as an iron replacement product for 4 weeks in rats showed that IONP can lead to iron accumulation in the ventricles. Iron concentration changes over time were quantified by the QSM technique. Slight changes in iron content in the striatum and corpus callosum were reported by using regions of interest (ROI) analysis, which may be related to iron deposition in the brain parenchyma. Also, the histopathological assessment showed choroid plexus hemosiderosis and midbrain vacuolation in the brain parenchyma [128].

In an in vivo study, radiolabeled aminopropyltriethox-ysilane(APTS)-coated IONPs were instilled intranasally in Sprague Dawley rats in a concentration of 10 μg （in 10 μl）. IONPs concentration in local areas on the seventh day of exposure was measured. The olfactory bulb, stria-tum, hippocampus, brain stem, cerebellum, and frontal cortex showed the highest concentration of IONP depositions, respectively. Even more than 50% of IONP remains in the striatum and hippocampus by 14 days later. Besides, oxidative damage increases in the stria-tum and hippocampus. Following in vivo study, toxicity mechanisms induced by IONP were investigated in dopaminergic neuronal PC12 cells. Incubated PC12 cells with IONPs (100 and 200 mg/ml) showed significant cytotoxicity including elevated MDA levels and a decrease in levels of GSH-PX and SOD. Exposed PC12 cells also showed an increase in phosphorylation of c-Jun, JNK, and p53, which were associated with oxidative stress and cell death [129]. To the best of our knowledge, there is no certain range of maximum permissible concentrations of IONPs in different areas of the brain. This varies for IONPs and depends on physicochemical properties and standardization.

IONPs surface coating

It is well known that optimizing the physicochemical parameters of IONPs is highly effective to minimize the interactions between these NPs and cells, immune response, and toxicity. Whenever a new nanoparticle is made, one of the first important things that need to be considered is its surface coating. The coating preserves the inner core of the nanoparticle and prevents the release of nanoparticles. However, the coating itself should not be toxic. One way to reduce the toxicity of nanoparticles is to coat them. Coating nanoparticles, in addition to making them viable and reducing their toxicity, also makes them more efficient [6]. Depending on the type and application of nanoparticles, different types of coatings have been used. Some coatings are used to protect nanoparticles from possible changes in the gastrointestinal tract, and some are used to conjugate mate-rials into nanoparticles. Nanoparticle coatings affect their absorption and biodistribution in the body and are even effective in the autophagy of nanoparticles [14, 108, 117]. Like most nanoparticles, IONPs contain an iron oxide core and a protective coating. The surface coating can optimize IONPs function and their cytotoxicity properties. Therefore, the surface coating seems essential for optimizing properties of IONPs including stability, biocompatibility, multi-functionalization, optimal biodegradation, hydrophilic interactions, and solubility [109]. The surface coating could be related to IONPs physicochemical characteristics including interactions with biological components, cellular uptake, in vivo fate, and toxicity. It also affects the fate and biological effects of IONPs. The coating provides an attachment layer to different molecular ligands such as chemical groups (e.g., carboxyl and hydroxyl) and biomolecules(e.g., peptides and polysaccharides), the so-called functionalization [6]. Because of colloidal instability of bare IONPs, several natural and synthetic surface coatings such as chitosan, dextran, citrate, Pluronic, polyethylene glycol (PEG), poly(ethylenimine)(PEI), polyvinyl alcohol (PVA), silica, and gold have been used. PEG is the most popular coating polymer because it prevents the aggregation and opsonization of nanoparticles. PEI is used to convey DNA/siRNA.In our studies, we have used dextran, a hydrophobic natural polymeric carbohydrate with a neutral charge [115, 130-134]. Although the proper coating can stabilize IONPs, avoid agglomeration, and prevent the dissolution and release of toxic ions, there are reports regarding the relative toxicity of surface-coated IONPs. In this regard, Kazemipour et al. reported that 100 mg/kg of IONPs coated by dextran induced a significant decrease in hepatic GSH level and CAT activity and a significant increase in hepatic MDA level of rats [135]. In a study Feng, et al. showed that PEI-coated IONPs caused severe cytotoxicity through multiple mechanisms such as ROS production and apoptosis. Whereas, PEGylated IONPs showed a slightly cytotoxic effect only at high concentrations. In addition, PEI-coated IONPs exhibited dose-dependent lethal toxicity in BALB/c mice [136]. The results of an in vitro study showed that magnetic nanoparticles coated with the shortest 0.75 kDa polyethylene oxide (PEO)tails caused cytotoxicity and there was an inverse correlation between the PEO tail block length with toxicity [137]. Badman and et al.examined the dose-dependent neurotoxicity of dextran-coated IONPs on cultured primary neurons and showed that concentration above 20 ug/ml increased cellular ROS and lead to cell death [138]. Therefore the presence of a strong iron chelator can improve the potential benefits of IONPs with different coating and prevents their possible toxicity of them. 

This article is extracted from Bardestani et al. J Nanobiotechnol (2021) 19:327 https://doi.org/10.1186/s12951-021-01059-0