**1. Introduction**

Biogenic iron is present in all biological systems. Detail regulation of iron metabolism was described previously [1–3]. Nanomaterials, including iron nanoparticles (NPs), are widely used in various industrial applications. However, the fast development of nanotechnologies and nanomaterials may pose a serious health hazard for humans and animals [4]. Ultrasmall superparamagnetic iron oxide nanoparticles of γ-Fe2O3 (maghemite) and Fe3O4 (magnetite) with the size of 10–50 nm (ultra-small superparamagnetic iron oxide nanoparticles (USPIONs)) can be used in various biomedical and medical applications [5–7]. The advantage of the USPIONs lies in the possibility to use them for targeted drug delivery in the presence of the magnetic field [8]. Stability of USPIONs depends on the local microcellular environment (chemical composition, pH, etc.). Intraendosomal degradation of nanoparticles poses a risk of iron overload, which may be dangerous mainly locally as they can modulate innate iron metabolism on systemic or cellular levels. There is an increasing number of studies that documented intracellular toxicity of iron NPs showing NP-induced inflammation, apoptosis, mitochondrial disorders and oxidative damage [9,10]. Recently, a correlation between exposure to iron oxide NPs and metabolism is of particular concern in nanotoxicology related fields, as NPs can potentially enter to iron metabolism and, thus, to affect its physiological roles. Iron NPs may also increase reactive oxygen species production and to produce oxidative stress, which can further induce adverse effects on DNA, proteins as well as membrane lipids [9,11] and to induce inflammation, changes in blood pressure (BP) regulatory systems via modulation of vascular function. Yet, there is still limited information on the uptake of the USPIONs to the individual organs and tissues and their possible effects on metabolism and physiological functions.

From the methodological point of view, iron content in the tissues can be determined using colorimetric, spectrophotometric, histochemical methods or by the technique of atomic absorption spectrometry depending on the purpose [12,13]. However, these methods do not allow to distinguish clearly between the biogenic iron and USPION-originating iron. Both biogenic nanoparticles (e.g., ferritin) and USPIONs are superparamagnetic, however, usually with the different blocking temperature [14,15].

SQUID magnetometry is a novel approach to quantify different iron forms in biological samples with high sensitivity that may provide new information for the investigation of iron NPs effects on living organism as well as for the understanding of the pathomechanisms of various diseased states. SQUID magnetometry is one of the methods enabling to determine the blocking temperature (e.g., so-called zero field cooled—ZFC, field cooled—FC and alternating current (AC) measurements) [16,17] and in such a way to identify the presence of applied USPIONs. In previous experimental research, SQUID magnetometry was widely used as a tool for determination of the various form of iron. Measurements were done using iron nanoparticles, namely in cell cultures [18–20], after in vivo treatment in mice [21,22] and also in embryos of Xenopus Laevis [23]. Using SQUID magnetometry, Janus et al. [24] showed that blood of patients with atherosclerosis was characterised by a higher concentration of ferrimagnetic particles such as Fe3O4 and γ-Fe2O3 (associated with the elevated values of the magnetic saturation (*M*s)) and significant changes in the superparamagnetic behaviour characterised with changes in the remnant magnetisation (*M*r) and the magnetic coercivity (*H*c).

The question of the measurement of biogenous iron content is also very important. SQUID magnetometric determination of biogenic iron was performed in various tissues of mice (duodenum, liver, spleen, kidney, heart or brain) [21,25] and rat blood [26]. In addition, iron content was also determined by SQUID magnetometry in human brain [27]. The authors of the above-mentioned studies used various techniques to characterise and determine the amount of iron. Measurement of the *M*(*H*) dependences is the standard method of iron determination by magnetometry, however, more information can be obtained by measurement of the temperature dependences of magnetisation, e.g., the ZFC and FC magnetisation characteristics. Another way is the determination of the isothermal remnant magnetisation (IRM) or using the AC susceptometry.

SQUID magnetometry, in combination with biomedical research, can provide a better understanding of iron metabolism in various diseased states as well as to distinguish biogenic iron from that originating from USPIONs. However, the investigation can be difficult, when NPs are used in very low doses which are diffusely distributed in the human or animal body.

Thus the aim of our study was to develop the method for determination of the amount of the USPION-originated iron in the tissues of rats and to investigate the magnetic characteristics of the liver, left heart ventricle, kidneys, aorta and blood of WKY rats after i.v. application of the low dose of USPIONs.
