**4.1. Magnetic iron oxide nanoparticle effect on normal cells**

Iron ions play major biological roles in different physiological processes, including DNA synthesis, oxygen transport, mitochondrial respiration, heme synthesis, and in metabolic functions at central nervous system level (nitric oxide metabolism, oxidative phosphorylation and myelin and neurotransmitter synthesis). Moreover, iron proved to be an essential factor for an appropriate function of neurons by acting as cofactor for tyrosine hydroxylase, an enzyme with a critical role in dopamine synthesis and the viability of neural cells. A dysregulation of the iron homeostasis or transport leads to unbalanced physiological functions and cytotoxic reactions. The free intracellular Fe2+ ions react with hydrogen peroxide (H2 O2 ) and determine the generation of reactive oxygen species (ROS), process known as Fenton reaction. An increased ROS concentration activates a cascade of events (release of iron ions into the cytosol by inducing an augmented permeability of the outer mitochondrial membrane and detrimental effects on lysosomal membrane; lipid peroxidation, damaged proteins, break of DNA chains and degradation of bases, mutations, deletions or translocations at nuclear level) that has as endpoint cell death. The pathologies that are associated with this type of cellular damage are aging, cancer and neurodegenerative diseases [91]. Another mechanism of inducing cell death by the iron ions is the apoptotic pathway via mitochondria, as follows: a high amount of iron ions into the mitochondria determine the opening of the mitochondrial transition pore, release of Ca2+ and cytochrome c and activation of apoptotic cascade [91, 92].

posed the use of different polymers like covering agents or surfactants like starch, heparin,

Polymer coating can be accomplished during or after the synthesis of magnetic nanoparticles. Polyethylene glycol (PEG) is a water-soluble, biocompatible hydrophilic polymer that can be used successfully in the synthesis of biocompatible nanoparticles with increased resistance to blood circulation [84]. Another alternative to covered magnetic nanoparticles is the use of copolymers that produce core-shell nanoparticles with possible applications in drug trans-

The use of inorganic compounds such as gold, silver, silica gel and carbon as surfactants not only provides good stability to the nanoparticles but also allows functionalizing their surface by grafting certain biological ligands. Covering of magnetic nanoparticles with gold seems to be ideal because of its low reactivity; however, coating the magnetic nanoparticles directly with gold is very difficult due to the different nature of the two surfaces [86–89]. The silica gel is the most widely used compound in the preparation of functionalized iron oxide nanoparticles surface, because it has several advantages: excellent biocompatibility, hydrophilicity, the feasibility of integrating other functional groups on the surface due to terminal silanol groups that can react with different coupling agents, provides good stabilization of the magnetic iron oxide nanoparticles in the solution, prevents the interaction between the nanoparticles thus preventing the agglomeration of the particles over time and ensures better encapsulation [90]. A very good coating of carbon layers provides an effective barrier against oxidation and acidic erosion of magnetic nanoparticles. It is therefore possible to synthesize carbon-coated magnetic nanoparticles that are thermally stable, biocompatible and also have high oxidation

Iron ions play major biological roles in different physiological processes, including DNA synthesis, oxygen transport, mitochondrial respiration, heme synthesis, and in metabolic functions at central nervous system level (nitric oxide metabolism, oxidative phosphorylation and myelin and neurotransmitter synthesis). Moreover, iron proved to be an essential factor for an appropriate function of neurons by acting as cofactor for tyrosine hydroxylase, an enzyme with a critical role in dopamine synthesis and the viability of neural cells. A dysregulation of the iron homeostasis or transport leads to unbalanced physiological functions and cyto-

mine the generation of reactive oxygen species (ROS), process known as Fenton reaction. An increased ROS concentration activates a cascade of events (release of iron ions into the cytosol by inducing an augmented permeability of the outer mitochondrial membrane and detrimental effects on lysosomal membrane; lipid peroxidation, damaged proteins, break of DNA chains and degradation of bases, mutations, deletions or translocations at nuclear level) that has as endpoint cell death. The pathologies that are associated with this type of cellular

O2

) and deter-

toxic reactions. The free intracellular Fe2+ ions react with hydrogen peroxide (H2

chitosan, dextran, oleic acid, polyethylene glycol (PEG), etc.

stability, which is crucial for certain applications [89].

**4.1. Magnetic iron oxide nanoparticle effect on normal cells**

**4.** *In vitro* **biological impact**

port (drug vector) [85].

242 Iron Ores and Iron Oxide Materials

Based on these data, concerning the toxicity induced by iron ions, it is imperative to study the possible toxic effects induced by M-IONPs, mainly since these particles present a higher reactivity as compared with the normal sized ones. The magnetic character of iron oxide nanoparticles offers some advantages, including the capacity of this nanosized compounds to be driven to targeted sites by an external magnetic field, even to tissues and organs that are difficult to reach in normal conditions (blood brain barrier and central nervous system). M-IONPs penetrate into the cells via receptor-mediated endocytosis and settle into the lysosomes, organelles characterized by the presence of an acidic medium, where it takes place the metabolization of the nanoparticles and free iron ions are released into the cell [91].

In a previous study, it was demonstrated that M-IONPs penetrate differentially into the neural cells (glial cells, primary neurons of the cerebellum, microglia, astrocytes, oligodendrocytes and Schwann cells), based on their dimensions: large size nanoparticles were absorbed by endocytosis, whereas small sized ones via pinocytosis [91, 93]. It was also proved that exposure to M-IONPs has an impact on iron homeostasis by upregulating the proteins responsible for iron storage or export from the cell and by downregulating the proteins expression involved in iron uptake [94].

Besides these positive features, application of an external magnetic field leads to accumulation of M-IONPs in target cells and potential toxicity. The accumulation of iron into the cells after exposure to M-IONPs seems to be dependent on several factors, such as (i) concentration and dose of M-IONPs (high concentrations require a longer period for elimination—several months, whereas M-IONPs in low concentrations can be eliminated within 3 weeks), size (small size nanoparticles cumulate in increased concentrations as compared to large size nanoparticles), shape (spherical nanoparticles present a longer degradation process due to a small contact surface), coating (some coating agents may prolong the degradation process or may increase it), the functional groups (the positively charged functional groups present in M-IONPs structure increase their uptake by the cells) and cell type (microglia have a higher affinity for M-IONPs, whereas into the brain endothelial cells penetrate less nanoparticles) [91].

A significant number of studies sustained that M-IONPs exerted *in vitro* and *in vivo* toxicity. The main players responsible for toxic effects are considered to be the iron ions released from M-IONPs at lysosomal level, which react with hydrogen peroxide and lead to ROS generation [91]. Exposure of neural cells to M-IONPs was associated with a low concentration of ROS, but a reduced level of glutathione and mitochondrial membrane hyperpolarization [95]. Other studies conducted on healthy cell lines (both human and animal origin) pointed out that bare M-IONPs may induce cytotoxic effects via ROS generation, leading to cell death [96–98].

The oxidation state of iron (Fe2+ or Fe3+) plays a major role in determining the nanoparticles toxicity according to the studies that affirm that Fe3+ in Fe2 O3 is more toxic than Fe2+ in Fe3 O4 and causes more DNA oxidation [91, 99].

**Figure 5.** The impact of magnetite and maghemite obtained by combustion method on HaCat cell morphology after 24 h stimulation.

**4.2. Magnetic iron oxide nanoparticles effect on cancer cells**

and apoptosis.

magnetism and (iii) presence of a coating agent [104].

to activate at cellular and molecular levels [105].

higher, but they also encounter macrophages in deeper compartments.

SPIONs (superparamagnetic iron oxide nanoparticles) are the most frequently used iron oxide nanoparticles in the biomedical applications due to their proper size (range between 50 and 200 nm) and the magnetic properties responsible for the lack of particle aggregates *in vivo.* Another type of iron oxide nanoparticles is represented by USPIONs (ultra-small superparamagnetic iron oxide nanoparticles), which have a diameter lower than 50 nm. The mandatory features of M-IONPs that must be analyzed in order to establish the bioavailability and the possible interactions with endogenous compounds (proteins, immune system cells, etc.) are: (i) size (the recommendable size for biomedical applications is between 10 and 200 nm; the ones that are too big will be assimilated by liver and spleen cells, the ones that are too small will be filtrated by the kidneys and their life in the bloodstream is reduced); (ii) superpara-

**Figure 6.** Mechanisms of toxicity induced by M-IONPs to normal cells (neural cells and hepatocytes): oxidative stress

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The affinity of liver, spleen, bone marrow and lymph nodes for SPIONs after their removal from the blood by the mononuclear phagocytic system (MPS) after intravenous administration represents the reason for the study of this type of nanoparticles as contrast agents but also for their use as delivery tools for chemotherapeutic agents. USPIONs due to their small size possess the capacity to escape macrophages of MPS surveillance and their circulation time is

The changes concerning the surface of the nanoparticles by using a coating agent proved to exert multiple roles: to improve colloids stability, to enhance the bioavailability and the bloodstream half-life and to reduce precipitation and formation of conglomerates [104, 105]. M-IONPs were used as drug delivery agents and as contrast agents based on their potential

The concentration of M-IONPs is also important in the assessment of M-IONPs toxicity. In one of our previous studies developed on HaCat cells (human keratinocytes), it was shown that concentrations lower than 25 μg/mL did not induce toxicity in terms of viability and cytoskeleton changes (**Figure 5**) [100].

Shelat and coworkers indicated a dose-dependent cytotoxic effect of M-IONPs on mouse embryonic fibroblast (NIH 3 T3) [101]. It was also assessed the effect of negatively charged superparamagnetic iron oxide nanoparticles on heart cells and no changes in actin cytoskeleton were observed, whereas in the case of brain and kidney cells, a disruption of the actin cytoskeleton was detected, but some increased vascular permeability was seen after exposure [102].

Another sign of toxicity that was described after neural cells exposure to M-IONPs was represented by protein aggregation. In addition, it was shown that M-IONPs induce apoptosis of hepatocytes in a mitochondrial-dependent way consisting of upregulation of pro-apoptotic markers (Bax and Bad) and downregulation of bcl-2 (anti-apoptotic); decrease of mitochondrial membrane potential followed by the release of cytochrome c into the cytosol what leads to activation of caspases cascade and apoptosis induction (**Figure 6**) [91].

Based on the data that were presented in this section, it could be said that the mechanisms involved in M-IONPs toxicity are accumulation of iron ions, oxidative damage by generating reactive oxygen species, protein aggregation and apoptosis.

Mutagenic effects of M-IONPs on different murine and mammalian normal cell lines were clearly synthesized in an extensive review [103].

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**Figure 6.** Mechanisms of toxicity induced by M-IONPs to normal cells (neural cells and hepatocytes): oxidative stress and apoptosis.
