Evaluation of Magnetosensitive Cytostatic Concentration and Different Mechanisms of their Antitumor Effects

The review covers different aspects of structural and functional features of magnetic nanoparticles. Especially those which are associated with their interaction with cells and cause development of oxidative stress, apoptosis disruption of DNA structure and cytoskeleton, changes in intracellular signal cascades etc. It is suggested that use of iron oxide nanoparticles for magnet-driven drug delivery in cancer therapy might be safe and promising because it can enhance antitumor effects of known cytostatic drugs.


INTRODUCTION
Magnetic materials based on iron, cobalt, nickel or their oxides are widely used in different fields of modern technologies [1]. Particularly, because small-sized magnetic nanoparticles (MNPs) demonstrate properties, which differ from those in macroscopic scale [2]. Nowadays MNPs are Review Article mainly used in biomedicine as MRI contrasting agents and for tumor treatment using local hyperthermia with alternating magnetic field [2][3][4]. Another promising application of MNPs is their use for target delivery of cytostatics to the tumor region using external static magnetic field (SMF) [5,6].

PHYSICAL CHARACTERISTICS OF MNPS
The main specific features of MNPs which determine their magnetic moment are shape, size and surface properties [2]. It is known that interaction between magnetic moments of atoms of the same material causes development of the particular magnetic structure. Reduction of MNPs size might cause the situation when every particle would carry only one magnetic domain, resulting in its totally different properties compared to the entire material [7]. MNPs are often characterized by superparamagnetic properties due to their small size. It means that vectors of every single MNP magnetic moments would rotate in random direction only due to particle thermal motion, resulting in zero total magnetic moment of entire MNPs [8].
Use of external magnetic field causes alignment of magnetic moment vector directions and significant increase of total magnetic moment of the material. Such features and absence of residual magnetization after treatment by external SMF make possible stabilization of MNPs in colloid solution without their agglomeration [4].
It is known that many factors affect MNPs morphology and the main among them are conditions of synthesis reactions [9]. It is accepted that the shape of MNPs greatly influence on their biodistribution, but detailed mechanisms of this process are not understood in detail [ After cellular uptake by endocytosis MNPs often form a cluster in lysosomes, where they degrade in presence of hydrolytic ferments at low pH to iron ions according to classical cellular iron metabolism pathways. This was proved by our light and electron microscopy studies in vitro on MCF-7 human breast cancer cells. Also we showed that cisplatin-resistant cells accumulated MNPs more actively compared to w/t MCF-7 cells [32]. It should be mentioned that application of 150 mT SMF significantly accelerated accumulation of MNPs, resulting in higher numbers of nanoparticles detected inside the cells.
Accumulation of different MNPs in cells often causes activation of ROS generation, which might serve as a defense mechanism to neutralize xenobiotics, as well as apoptosis inducers [36,37]. The degree of toxic side effects depends on the type of studied cells, but many of them show activation of defense mechanisms able to neutralize low amounts of ROS, making only high concentrations of MNPs dangerous [23,38]. Induction of ROS generation by MNPs often depends on their coating composition as well as on MNPs concentration inside the cell [39]. For example, nickel ferrite MNPs were shown to induce toxic effects in cells by activation of ROS generation, which depended on high cellular concentration of nanoparticles [34]. The same results about activation of ROS generation by iron oxide MNPs, especially in presence of exogenous SMF, we obtained on MCF-7 and Ehrlich ascetic carcinoma cells in vitro and in vivo.
It is thought that iron oxide MNPs cause Fentontype chemical reactions, which lead to active ROS generation. It was shown that naked magnetite nanoparticles had were characterized by severe cytotoxic effects [40]. At the same time, cytotoxic action of maghemite (fully oxidized iron oxide, Fe 2 O 3 ) is not associated with Fenton reactions [7]. In general, mechanisms underlying ROS generation by MNPs are not fully understood, but there is a hypothesis that changes in structured electron configuration of the nanoparticle surface lead to development of new electron donor or acceptor sites, resulting in ROS generation. MNP-induced ROS generation activates defense antioxidant systems in multistage process through transcription factor Nrf-2 [41], resulting in elevation of more than 200 phase II antioxidant proteins expression (haemoxygenase-1, superoxide dismutase, etc.). As the damage increases, defense systems are substituted by МАРК-and NF-kB-activated intracellular signal transduction pathways, leading to excretion of pro-inflammatory cytokines, chemokines and matrix metalloproteinases (MMPs), and, finally, to apoptosis [34, 36,42]. Such activation of МАРК cascades was detected in cells of respiratory and gastrointestinal tracts, blood cells, skin and neurons. We also found some changes in expression of apoptosis regulator proteins (p53, Bcl-2 and Bax), as well as miRNA expression profile in MCF-7 cells with different sensitivity to cisplatin, which confirmed increase of cell number in apoptosis after their treatment with stabilized iron oxide MNPs [43]. It should be mentioned that complex of MNPs with cisplatin resulted in much more serious effects, which were amplified by SMF.
Recent studies show direct correlations between MNP size, shape and dispersion properties with cytotoxic effects and pro-inflammatory cell reaction [10]. For example, ROS control MMP activity via two different mechanisms: МАРКinduced overexpression of ММР genes and direct oxidation of thiol groups in pro-MMP molecules, resulting in their activation [44]. So, MMPs might serve as a messenger in the process of macrophage activation in presence of MNPs. It was shown that accumulation of chitozan-coated MNPs led to increase invasion potential of cells, caused by MMPs activation.
Size of MNPs and features of their intracellular accumulation also affect their interference with cytoskeleton elements [45]. Interaction between MNPs and cytoskeleton proteins might be direct (MNPs reached cytoplasm) or indirect (MNPs localized in endosomes). The last type of interaction is most commonly observed [46]. It is suggested that different coating types of MNPs cause different changes in cytoskeleton, while high concentration of uptaken particles also causes its disruption. It is also known that cytoskeleton takes part in many intracellular signal cascades, so, one of the main goals of studies is to found whether MNP-induced cytoskeleton disruptions are able to cause secondary effects like cell death, changes in proliferative activity etc [7].
It is known that regulation of many cellular functions, including cell growth, motility, and differentiation is highly dependent on cellular adhesion properties. Research data show that cellular adhesion properties could be interfered by MNPs, but results of these studies are controversial. For example, ZnO-containing composite caused significant reduction of astrocyte adhesion properties after 4 hours of incubation, and after 72 hours of incubation this index was almost 2 times lower compared to control. This might be a result of changes in adhesion receptors expression on cell surface induced by nanomaterials [47,48]. We showed that MNPs can significantly change adhesion properties and colony-forming activity of MCF-7 cells, resulting in reduction of their invasion properties and proliferation activity [15].
In another study endotheliocytes were cocultivated with non-toxic iron-oxide MNPs concentrations for 24 hours and showed no changes in their adhesion properties. Authors did not found significant changes in cell morphology and MNPs aggregation features [40].
Intracellular signal pathways might be changed not only due to cytoskeleton disruptions under MNP impact, but also because of different other mechanisms, such as: (1) genotoxic effects, caused by high ROS levels, (2) changes in gene and protein expression as a result of disruptions in transcription and translation processes, (3) changes of gene or protein expression pattern due to increase of metal ion levels, (4) changes in protein activation by preventing their interaction with stimulating factors, e.g. cell surface receptors, (5) [52].
Biodegradation of MNPs is the mechanism, which results in formation of free trivalent iron ions after solution of the MNP core [30]. As it was mentioned, kinetics of MNP solution depend on their surface coating. Accumulation of free trivalent iron ions might in some cases result in generation of high levels of ROS, inducing apoptosis or inflammation.
Another possible mechanism of MNP toxicity is their interaction with biological molecules. They are able to aggregate with serum proteins due to their charge if their coating is unable to prevent this process [53]. The use of MNPs for local hyperthermia or for target drug delivery cause new problems, which also should be taken into account. Hyperthermia needs alternating magnetic fields, which are used to increase MNPs temperature and kill tumor cells. It is known that alternating magnetic fields can also damage healthy tissues, which are situated near the tumor burden. Magnet-driven target delivery of drugs or MRI contrasting with SMF, is thought not to cause direct effects on cells [25], but we already showed in vitro that even 150 mT SMF alone starting from 3 hours of continuous impact was able to cause significant changes in MCF-7 and Ehrlich ascetic carcinoma cells, resulting in ROS generation, genotoxic effects, changes in mitochondria activity and in accumulation of MNPs in cells.
In the last case toxic effects might be associated with active income of MNPs into cells and with changes in localization of endosomes inside the cell and their malfunctioning [24]. So, endocytosis is the main mechanism of MNPs uptake by cells (Fig. 1). Then nanopartcles degrade in lysosomes to iron ions, resulting in generation of ROS due to Fenton-type reactions or accumulate in cytoplasm and nucleus, causing changes in cytoskeleton, mitochondria and genotoxic effects. Accumulation of these changes usually lead to cell death due to apoptosis.

PERSPECTIVES OF MNPS USE FOR TARGET DELIVERY OF ANTITUMOR DRUGS WITH MAGNETIC FIELD
Antitumor therapy is often based on use of chemotherapeutic drugs, which are highly cytotoxic, but have low specificity against their biological target [54]. Usually they cause severe systemic damage to the organism, resulting in well-known side effects due to interaction between antitumor drugs and healthy tissues [55].
The idea of using SMF (SMF implants or external SMF) as a vector to increase drug accumulation in tumor region first appeared in early 1980s. Widder et al. [56] performed first preclinical studies with use of magnetic microspheres, covered by albumin, which contained doxorubicin, for treatment of transplanted rat tumors.
The main advantage of target drug delivery by MNPs and SMF is increase of local cytostatic concentration in the tumor region with use of lower doses of drugs [57]. One of the complex problems in this approach is small size of MNPs. On the one hand, this prolongs their circulation after injection, increasing chances of successful drug delivery to the tumor with use of external SMF. On the other hand, small diameter of MNPs is a limiting factor for use of SMF [58], because magnetic force value depends on MNP magnetic moment and magnetic field gradient. This force is proportional to the MNP volume, meaning that 10 times decrease of linear MNPs dimensions would result in 1000 times decrease of force value. It was shown that minimal diameter for agglomerates with phospholipid coating which were effectively driven by SMF was 40 nm, for MNPs with polymer coating -70 nm. Such difference is observed because of bigger volume of magnetite in MNPs, covered with phospholipids [59,60].
Motion of MNPs in matrix or in fluid depends on many factors (magnetic field gradient, temperature, viscosity of the substance, velocity of fluid flow and interaction of MNPs with fluid components, size and shape of MNPs). Nowadays dynamics of MNP motion in bloodstream is actively studied [61]. Magnetic field gradient is needed for accumulation of MNPs in particular region, because in homogenous fields the value of magnetic forces applied to MNPs would be equal to zero. Magnetic field gradient must be strong enough to overcome bloodstream, so the closer magnet would be to the vessel wall, the better the resulting effect would be [12,62]. It should be mentioned that MNPs would also accumulate in tissues which lie between target and SMF source. So, external magnets are likely to be used when target region is located near the body surface. Together with our colleagues we developed different systems of static magnets, allowing us to create a high-gradient magnetic field in the tumor zone (induction near pole 0.6 T; gradient 40 T/m), which made us possible to effectively accumulate MNPs in rat tumor tissue (Guerin carcinoma, Walker-256 carcinosarcoma) and achieve better therapeutic effects without elevation of their general toxicity [5].
Scientists showed significant increase of dextrane-coated MNPs penetration rate through artificial three-layer membrane under 0.410 T external SMF [63]. In other study Lamkowsky et al. found that brain astrocytes accumulate iron oxide MNPs covered with dimercaptosuccinate and this process depended on duration, temperature and MNP concentration. MNP accumulation rate proportionally increased with magnification of external SMF induction, resulting in growth of cellular iron content from 10 nmol/mg of protein after 4 hours of incubation at 37°С to 12000 nmol/mg of protein [64]. The mentioned data suggest that use of external SMF enforces interactions of iron MNPs with cell membranes as well as their uptake by astrocytes in vitro. In vivo (on Guerin carcinoma) we observed enhancement of cisplatin and MNP-cisplatin nanocomposite cytotoxic action by SMF. We found significant increase of necrosis and apoptosis rates in tumor tissue. Many Guerin carcinoma cells showed changes in normal structure of cypolasm organelles and had iron oxide nanoparticles in cytoplasm or nuclei. Aggregates of iron oxide MNPs were the biggest after the combined impact of MNP-cisplatin nanocomposite and SMF. Another feature of SMF impact was damage blood vessels endothelium, which resulting in elevation of their permeability, thus also increasing the antitumor effect of the chemotherapeutic drugs [5].

CONCLUSION
So, during last years many promising MNP models were developed and they proved their efficacy in vitro and in vivo.