Low Doses of Polyethylene Glycol Coated Iron Oxide Nanoparticles

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Low Doses of Polyethylene Glycol Coated Iron Oxide Nanoparticles Cause Significant Elemental Changes Within the Main Organs Agnieszka Skoczeń, Katarzyna Matusiak, Zuzanna Setkowicz, Aldona KubalaKuku#, Ilona Stabrawa, Ma#gorzata Ciarach, Krzysztof Janeczko, and Joanna Chwiej Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00110 • Publication Date (Web): 02 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Chemical Research in Toxicology

Low Doses of Polyethylene Glycol Coated Iron Oxide Nanoparticles Cause Significant Elemental Changes Within the Main Organs Agnieszka Skoczeń1*; Katarzyna Matusiak1; Zuzanna Setkowicz2; Aldona Kubala-Kukuś3,4; Ilona Stabrawa3,4; Małgorzata Ciarach2; Krzysztof Janeczko2; Joanna Chwiej1. 1

AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Department of Medical Physics and Biophysics, Krakow, Poland 2 Jagiellonian University, Institute of Zoology and Biomedical Research, Department of Neuroanatomy, Krakow, Poland 3 Jan Kochanowski University, Institute of Physics, Kielce, Poland 4 Holy Cross Cancer Center, Kielce, Poland * Corresponding author address: [email protected]

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Table of Contents

Abstract The main goal of this study was to evaluate the elemental changes occurring in the main rat organs (kidneys, spleen, heart, brain) as a result of PEG-coated magnetic iron oxide nanoparticles (PEG-IONPs) administration. For this purpose twenty four animals were divided into four equinumerous groups and the three of them were intravenously injected with PEG-IONPs dispersed in 15% solution of mannitol in dose of 0.03 mg of Fe per 1 kg of the body weight. The organs were collected 2 hours, 24 hours and 7 days passing from NPs administration respectively for the 2H, 24H and 7D experimental groups. The forth group of animals, namely control group, was injected with 1 ml of physiological saline solution. For the analysis of subtle elemental changes occurring in the organs after nanoparticles injection, highly sensitive method of total reflection X-ray fluorescence spectroscopy was used. Obtained results showed that administration of even such low doses of PEGIONPs may led to statistically significant changes in the accumulation of selected elements within kidneys and heart. Two hours and seven days from NPs injection the Fe level in kidneys was higher comparing to control rats. Elevated levels of Cu, possibly associated with systemic action of ceruloplasmine enzyme, were found within kidneys in 24H and 7D groups, whilst in heart the similar observation was done only for 24H group. The levels of Ca and Zn increased in kidneys and heart during the first two hours from the injection and were again elevated in these organs 7 days later. The abnormalities in Ca and Zn accumulations occurring exactly in the same manner may suggest that these elements may interplay either in the mechanisms responsible for the detoxification of the PEG-IONPs or pathological processes occurring as a result of their action. Key words: iron oxide nanoparticles, nanotoxicity, elemental anomalies, total reflection X-ray fluorescence, in vivo studies

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Introduction In the recent decades, the great progress in the development of new nanomaterials (NMs) dedicated for various fields of life and science has been observed. Their precise definition was given by the European Commission in the recommendation from 18th October 2011. Accordingly, the term ‘nanomaterials’ specifies any natural, incidental or manufactured material containing particles, in an unbound, aggregated or agglomerated state, where at least one of external dimensions, for 50% or more of the particles in the number size distribution, is in the size ranges 1-100 nm 1. In turn, nanoparticles (NPs) are most commonly defined as the objects with the diameter between 1 and 100 nm 2. NMs possess unique electrical, chemical, structural, and magnetic properties that makes them highly attractive especially for biomedical applications 3–5. The greatest potential to revolutionize clinical methods of diagnosis and therapy is attributed to magnetic nanoparticles (MNPs). The magnetic properties of these particular NPs cause that they can be used as a contrast agents for magnetic resonance imaging (MRI) and they can also serve as a targeted drug carriers enabling monitoring of their therapeutics distribution. Furthermore, MNPs can induce local hyperthermia in response to an external magnetic field and therefore selectively destroy cancer cells 6–9. Due to their small sizes, comparable with cellular components and biological molecules, NPs can easily exceed the natural barriers protecting the organism and induce adverse health effects 10,11. Moreover, because of the complexity of in vivo systems, interaction of nanoparticles with the components such as cells or proteins may result in unique biodistribution, clearance, immune response and metabolism 12. Therefore, the increasing attention is paid to determine the potential risk of engineered NPs. In spite of a numerous publications concerning the subject of nanotoxicity, the health and safety aspects of nanotechnology are still far behind its development. In particular, the impact of NMs on biological systems and the relationship between their properties and toxic biological response need to be thoroughly investigated 10,12,13. In turn, evaluation of the biodistribution and pharmacokinetics of NPs after administration to the body is crucial to enhance their functionality 12–14. Currently, most studies concerning the toxic effects of nanoparticles utilize cell culture models. While in vitro studies have been widely performed, there is still a great need for research on NPs behavior and potential adverse effects under in vivo conditions 12,15. Especially since the inconsistency between in vitro and in vivo effects of selected nanoparticles has recently been demonstrated 12,16. The essential element of the in vivo studies are those with animal use, which allow to evaluate the systemic impact of NPs on a living organism 12,15,17. Among different advantages of MNPs, predisposing them for the use in various medical applications, predominant attention is focused on biocompatible, biodegradable and easy to synthetize iron oxide nanoparticles (IONPs) 18,19. So far, the in vivo studies concerning IONPs toxicity have been focused on exposure to relatively high doses of NPs, which were equal or much higher than these used clinically in humans 6,18,20–22. In particular, Resovist, the drug clinically used in MRI diagnostics of liver, is administered at dose of 0.49 mg of Fe per kg of body mass 14,23. In turn, for Rienso, applied in anemia treatment, the injected dose is 12 mg of Fe per kg of body mass 24. To improve the nanoparticles biocompatibility and stability in the solution it is necessary to apply appropriate coating. These properties are attributed to the commonly used compound, which is polyethylene glycol (PEG) 27. Due to its hydrophobic properties, PEG prevents coated nanoobjects from opsonization by proteins present within circulatory systems, leading to the inhibition of the immune response and capturing of the nanoparticles by the mononuclear phagocytic system present inter alia within spleen 28,29. Such property increases nanoparticles circulation time and internalization efficiency, and thus makes PEG especially attractive coating for biomedical applications 27,30. In our last paper published in Nanotoxicology, the influence of much lower dose (0.03 mg Fe/kg of a body mass) of PEG-coated IONPs, suspended in 15% mannitol solution, on the elemental composition of rat liver was examined 25. The obtained results proved that even such small quantities of intravenously injected IONPs might induce changes in the systemic iron metabolism as well as in the liver contents of 3



the elements such as Cu, Ca and Zn 25. This paper is the continuation of the mentioned investigation and aims at evaluation of potential toxic effects of the same IONPs dose on the other rat organs, namely on kidneys, spleen, heart and brain. Existing reports suggest that IONPs may accumulate within the aforementioned organs, causing some transient histopathological and functional changes 14,15,18. Apart from that, the choice of the tissues was dictated mainly by functions played by these organs in the organism. For example kidneys, as a crucial element of body clearance, are desirable pathway for NPs removal from the body 14,26. Spleen in turn, together with previously considered liver, is important for immune response and forming mononuclear phagocytic system (MPS), which is responsible for IONPs clearance from the bloodstream 14. The possible impact of intravenously injected IONPs on heart, the central element of cardiovascular system, may be connected with its exposition to high blood flows 15,26. In this work the temporary and long-term influence of PEG-IONPs on the contents of Fe and other important elements, including Cu, Ca and Zn within selected organs were examined. The elemental anomalies within kidneys, spleen, heart and brain were assessed by comparative analysis of their concentrations quantified for animals subjected to NPs and controls. To achieve this goal the modern and highly sensitive analytical technique of total reflection X-ray fluorescence (TXRF) spectroscopy was used. With low detection limits (ppm-ppb range) TXRF method enabled us to examine the changes in Fe, Cu, Ca and Zn levels occurring in the mentioned organs after administration of relatively low dose of IONPs. Analyzing the tissues taken from animals at different times passing from NPs injection we were able to follow the dynamics of changes in their organ accumulations.

Experimental Procedures IONPs solution The mannitol (Baxter) solution of PEG-coated magnetic iron (II, III) oxide nanoparticles (Sigma-Aldrich 747408) was used in the experiment. The detailed protocol of solution preparation was placed in our previous publication 25. The hydrodynamic diameter and zeta potential of IONPs, evaluated with the dynamic light scattering (DLS) method at the wavelength of 633 nm, were equal to 35 nm and -98 mV respectively. The final concentration of Fe in the NPs solution was determined using S2 PICOFOXTM spectrometer (Bruker) and was equal to 8.14 ppm (standard deviation of the concentration was 0.86 ppm). Experimental animals and sample preparation Adult male Wistar rats came from the animal colony of the Department of Neuroanatomy (Institute of Zoology and Biomedical Research, Jagiellonian University, Krakow). All procedures involving animals were approved by the Bioethical Commission of the Jagiellonian University, Krakow, Poland (agreement no. 121/2015) and were performed in accordance with international standards. On the 60th day of the postnatal life, 24 animals were divided into two groups. One of them included 18 rats which were intravenously injected with 1ml of the IONPs solution. In order to minimize the influence of the injection on the obtained results the second control group (N) was treated with the same volume of physiological saline solution. After two hours, twenty four hours and seven days, counting from IONPs administration, animals originating from groups 2H, 24H and 7D (respectively) were weighted and perfused intracardially with 0.9% saline of high analytical purity in order to remove blood from their bodies. The kidneys, spleen, heart and brain were taken from each rat and deeply frozen in liquid nitrogen. Afterwards, the organs were separately packed and stored in the temperature of -80°C till the microwave-assisted digestion. In the next step, the whole organs were subjected to the microwave assisted digestion process performed in the mineralizer SpeedWave 4 (Berghof). The 0.3 ml of Ga internal standard in the concentration of 10 ppm was added to 1 ml of each digested sample and the mixture was homogenized. Afterwards, 2 µl of the solution was placed onto the special quartz glass carrier and dried on the heating plate. The detailed procedures of sample preparation were presented elsewhere 25. 4


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Apparatus and measurement conditions The measurements were carried out in the Laboratory of X-ray Methods, Institute of Physics at The Jan Kochanowski University in Kielce. The multielemental analysis based on the TXRF method was performed with S2 PICOFOXTM automatic spectrometer. The spectrometer is equipped with metalceramic air-cooled X-ray tube with molybdenum anode and multilayer monochromator. The target was set at the angle of 6° and the focal spot size was 1.2 x 0.1 mm2. The energy of the exciting beam was 17.5 keV. 25 quartz glass carriers with the samples were placed in the cassette and the measurement time of each sample was set to 20 minutes. Statistical analysis The Mann Whitney U test was applied for statistical evaluation of changes in elemental composition of the examined organs after IONPs injection. The non-parametric statistical test was applied as our data might not meet the assumptions concerning the normality, homoscedasticity and linearity (the population is too small to verify these assumptions) which are necessary for the use of parametric test. The differences were tested at the significance level of 5%. The statistical analysis was performed using the STATISTICA software (version 7.1). Elemental analysis Qualitative and quantitative elemental analysis was carried out with the use of TXRF spectroscopy. In the Figure 1, the exemplary TXRF spectra recorded for kidneys, spleen, heart and brain taken from selected control animal are presented. It is necessary to mention, that the spectral data acquisition as well as their fitting were performed with the Picofox Spectra 7 software.

Figure 1 The comparison of the exemplary TXRF spectra obtained for examined organs from selected control rat. Emission K-α lines originating from the analyzed elements (Fe, Cu, Ca, Zn) and used for further quantifications were marked with the black squares. The Ga was used as the internal standard. For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.

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According to the Figure 1, the following elements were detected within the all examined organs: P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, Pb, Br, Se and Rb. However, only Fe, Cu, Ca and Zn were considered for further quantifications. It was due to the fact that light elements such as P, S, Cl and K can be lost during wet digestion process whilst the concentrations of Mn, Pb, Br, Se and Rb were below the detection limits for some of the examined samples. In turn, the Si and Ar lines in the spectra originated from sample carrier and air respectively. Elemental concentrations calculation The concentration of the element in the measured liquid sample was calculated as follows: ∙ = (1) ∙ where: − concentration of the internal standard (Ga) in the liquid sample [ppm], − net pulse number for the internal standard in the sample spectrum [a.u.], − net pulse number for the element i in the sample spectrum [a.u.], − relative sensitivity for the element i. To calculate the final elemental concentration within organ the sample dilution factor and the liquid to organ mass conversion factor were considered, as it was described in our previous work 25: = ∙ ∙ (2) where: − the elemental concentration in the organ [ppm], − the elemental concentration in the diluted sample [ppm], − dilution coefficient (1.3 for each organ) [a.u.], – liquid to organ mass conversion factor for organ [a.u.].

Results Elemental changes occurring in selected rat organs after IONPs administration The TXRF spectroscopy allowed us to evaluate concentrations of Fe, Cu, Ca and Zn within the main organs taken from animals subjected to the PEG-IONPs and controls. In order to compare the data obtained for different experimental groups, the median contents of the elements in the examined tissues were calculated and are presented in Tables S1-S4 of Supporting Information for the Publication. Additionally, in the Figures 2-5, the median, minimal and maximal concentrations of Fe, Cu, Ca and Zn (respectively) together with the interquartile ranges recorded for 2H, 24H and 7D animals as well as control rats are shown as the box-and-whiskers charts. The statistical evaluation of the differences in elemental concentrations between IONPs injected and control groups was performed with nonparametric Mann-Whitney U test. The statistically significant increases (none decreases were detected) in the levels of examined elements in animals treated with IONPs are marked in box-and-whiskers charts. Additionally, the dynamics of elemental changes occurring within examined organs are schematically presented there. It was done by the evaluation of the statistically significant differences in Fe, Cu, Ca and Zn levels between subsequent moments after NPs injection. The arrows at the plots point at the direction of the observed changes (arrow sloping up – increase, arrow sloping down – decrease), whilst ‘+’ or ‘–’ mean the values higher or lower (respectively) comparing to controls. As one can see in the Figures 2-5, PEG-IONPs administration to animals may led to both temporary and long-term changes in the concentrations of the examined elements within the analyzed rat organs. 6


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The evaluation of changes of Fe content As on can see in the Figure 2, kidneys were the only organ for which the statistically significant anomalies in Fe concentration were observed. Two hours after NPs injection, the accumulation of Fe in kidneys was significantly higher than in controls but in the next 22 hours its content decreased to the normal level. However, on the 7th day from the injection the level of Fe relevantly exceeded the normal concentration again. None statistically significant anomalies in Fe concentration were observed for brain, heart and spleen. Nonetheless, as it can be seen from the plot showing the dynamics of changes in Fe levels, its concentration within spleen, similarly as in kidneys, tended to increase between 1st and 7th day from IONPs injection. Despite that, Fe content in 7D group did not exceed the normal level which indicates the slower nature of the changes in this element concentration for spleen comparing to kidneys.

Figure 2 The median, minimal and maximal values as well as lower and upper quartiles range of Fe concentration in the examined rat organs obtained for N, 2H, 24H and 7D groups. The p-values of Mann-Whitney U test for statistically significant differences between IONPs treated and control rats are placed on the charts. The statistically significant increases (p-value less than 5%) of Fe concentration with respect to N group are marked with regular up-arrows. Additionally, the dynamics of changes in Fe concentrations (statistically significant differences between subsequent moments after IONPs administration) is presented.

The evaluation of changes in Cu accumulation The differences in Cu concentrations between examined groups of rats are presented in the Figure 3. As one can see, 24 hours and 7 days from IONPs administration, the levels of Cu in kidneys taken from NPs treated animals were significantly higher than in control rats. In case of heart, similar relation was found only for 24H group. However, it was not statistically relevant at the assumed significance level of 5%. Neither statistically significant differences comparing to controls nor trends, were noticed for brain and spleen of animals subjected to NPs action. The changes of Cu concentration in kidneys and heart may be a result of ceruloplasmin enzyme release from the liver to the blood stream as it was described in our previous work 25. Accordingly, the elevated level of Cu was found within the blood serum 24 hours and 7 days after administration of PEG-IONPs. To 7



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make easier the interpretation of possible relations between the organ and serum levels of Cu, the dynamics of the changes in the serum concentration of the element is added in the Figure 3.

Figure 3 The median, minimal and maximal values as well as lower and upper quartiles range of Cu concentration in rat organs taken from N, 2H, 24H and 7D groups. The p-values of Mann-Whitney U test for statistically significant differences between IONPs treated and control rats and for trends are placed on the charts. The statistically significant increases (p-value less than 5%) of Cu concentration with respect to N group are marked with regular up-arrows whilst the upward trend (p-value between 5 and 10%) with dotted up-arrow. Additionally, the dynamics of changes in Cu accumulations is presented for selected organs and blood serum. Statistically significant difference between subsequent moments after IONPs administration is marked with regular sloping arrow whilst trend with dotted sloping arrow.

As one can notice from the plot showing the dynamics of changes in Cu levels, the concentrations of the element within heart and blood serum presented very similar pattern of variations. The content of Cu within the heart increased significantly between 2nd and 24th hour from NPs injection and this occurred in parallel with the elevation (trend with p-value between 5 and 10%) of Cu concentration in the serum. Both for heart and blood serum, Cu levels did not change during the first and the third observation periods. Although, 24 hours and 7 days from administration of IONPs the element level within kidneys was significantly higher in NPs injected animals comparing to control rats, the statistically relevant increase in Cu concentration did not occur during any of the three observation periods. Such result may suggest that changes in Cu level within the organ progressed much slower than within heart or blood serum. Furthermore, the accumulations of Cu within spleen and brain remained stable during all the observation periods. The evaluation of Ca and Zn concentrations As one can see from Figures 4 and 5, the differences in concentrations of Ca and Zn occurring between NPs treated and normal rats presented very similar pattern. The levels of both elements were significantly elevated within kidney 2 hours and 7 days from NPs injection. Similar relation was observed for heart but it was statistically significant only for Zn. In turn Ca accumulation presented explicit upward trend both 2 hours and 7 days after subjection to the NPs. Also the dynamics of changes in Ca and Zn contents exhibited very similar pattern. The increase of the concentrations of both elements within the organs occurred only during the first two hours from NPs administration.

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None differences in Ca and Zn concentrations were observed for brain and spleen of rats subjected to NPs action comparing to controls. Furthermore, for both organs, the levels of the elements remained stable between the subsequent times passing from the IONPs injection.

Figure 4 The median, minimal and maximal values as well as lower and upper quartiles range of Ca concentration in rat organs obtained for N, 2H, 24H and 7D groups. The p-values of Mann-Whitney U test for statistically significant differences between IONPs treated and control rats and for trends are placed on the charts. The statistically significant increases (significance level of 5%) of Ca concentration with respect to N group are marked with regular up-arrows whilst the upward trends (p-value between 5 and 10%) with dotted up-arrows. Additionally, the dynamics of changes in Ca concentrations is presented for selected organs. Statistically significant difference between subsequent moments after IONPs administration is marked with regular sloping arrow whilst trend with dotted sloping arrow.

Figure 5 The median, minimal and maximal values as well as lower and upper quartiles range of Zn concentration in rat organs obtained for N, 2H, 24H and 7D groups. The p-values of Mann-Whitney U test for statistically significant differences between 9 IONPs treated and control rats are placed on the charts. The statistically significant increases (significance level of 5%) of Zn accumulation with respect to N group are marked with regular up-arrows. Additionally, the dynamics of changes in Zn concentrations (statistically significant differences between subsequent moments after IONPs administration) is presented.



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Discussion In this paper, the elemental anomalies occurring in the main rat organs (kidneys, spleen, heart, brain) as a result of PEG-coated magnetic iron oxide nanoparticles administration were examined. To achieve this goal 24 animals were divided into four equal groups and three of them (2H, 24H and 7D) were intravenously injected with PEG-IONPs dispersed in 15% solution of mannitol in dose of 0.03 mg of Fe per 1 kg of the body weight. The applied dose was about 16 times lower than diagnostic dose of Resovist and about 425 times lower than therapeutic doses of Rienso used clinically in humans 23,24,31. To evaluate the changes in elemental accumulation a highly sensitive analytical technique of total reflection X-ray fluorescence spectroscopy was used. Among many advantages of the TXRF method, small amount of sample needed to spectral analysis and relatively low detection limits (ppm-ppb) make it especially suitable for detecting very subtle anomalies in elemental concentrations within biomedical samples 32–35. The high sensitivity of the TXRF technique results from the significant background reduction achieved by the measurement geometry of the method in which the primary beam does not penetrate the reflector but is almost entirely reflected from the sample surface. Hence also, the sample is excited twice (once by primary beam and next by the beam reflected from the carrier) thereby enhancing the intensity of the detected fluorescent radiation as well as signal to noise ratio (SNR) comparing to standard X-ray fluorescence (XRF) 35. In the following study, the TXRF method was applied to evaluate the changes in Fe, Cu, Ca and Zn concentrations occurring within the kidneys, spleen, heart and brain of rats subjected to intravenous injection of the PEG-IONPs. Analysis of the tissues taken from animals at different time intervals from NPs injection allowed tracking the dynamics of elemental anomalies resulting from the nanoparticles systemic action. The core material of nanoparticles used in the present study is magnetite (Fe3O4). This particular iron (II, III) oxide is the mixture of ferrous Fe3+ and ferric Fe2+ ions which may participate in the formation of reactive oxygen species (ROS) 36,37. Fe2+ ions are able to catalyze the hydroxyl radical’s (OH•) formation process from hydrogen peroxide (H2O2) through the Fenton’s reaction. In turn ferrous ions, in the presence of superoxide (O2•-) or other Fe3+ reducing agents, are reduced to Fe2+ form 38. This feature predisposes IONPs to induce cytotoxic effects such as an oxidative stress leading to damage within the organs in which they accumulate 39,40. The key role in the prevention from Fe induced oxidative stress plays ceruloplasmine enzyme which as an antioxidant inhibits free radical formation through the catalysis of Fe2+ to Fe3+ oxidation reaction 36,37,41. Obtained results revealed elevated Fe level within the kidneys after 2 hours and 7 days from IONPs injection. Rat kidneys are able to filter selectively the particles with the hydrodynamic diameter (dH) below 15 nm. The dH of the IONPs used in the presented study was equal to around 35 nm and they could not be removed from the body through the renal clearance but might accumulate in kidneys leading to the elevated Fe level 14,26,42. The decrease in the Fe concentration, that was observed in the organs between 2nd and 24th hour from the NPs administration may indicate at occurrence of nanoparticles redistribution from kidneys to the blood circulation 14,26,43. This conclusion seems to be confirmed by the gradual increase in Fe level within spleen between 1st and 7th day from subjection to NPs, which is probably connected with filtering of the redistributed IONPs from the bloodstream through the endocytosis in macrophages residing within the reticular meshwork of the organ 14,44. Nearly 95% of Cu within the serum is in the form associated with ceruloplasmin 36,45. As it was mentioned, this enzyme is essential for catalyzing Fe2+ oxidation to Fe+3, which enables Fe binding to the transferrin and its transport from the circulation to the cells via the transferrin receptor thereby preventing from Fe overload in serum 36,37,41. It has also been shown that ceruloplasmin demonstrates antioxidant functions against a wide range of compounds. It is believed to be responsible for about 80% of plasma antioxidant properties 41. What is more, ceruloplasmin is classified to positive acute phase proteins group, since its production increases due to infection and inflammation, which may be related to the response of 10


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the organism to the oxidative stress accompanying these processes 46,47. Other important enzyme of Cudependent activity is Cu,Zn-superoxide dismutase (Cu,Zn-SOD), which is the part of the cellular antioxidant protection network 48,49. Elevated concentration of Cu was observed within the kidneys after 24 hours and 7 days from IONPs injection. Such effect the most probably results from the increase of Cu level in the serum as a consequence of ceruloplasmin release from the liver to the blood stream what can be noticed from the dynamics chart in the Figure 3. Observed changes in Cu concentration after 24 hours from the NPs administration seem to follow organism reaction on enhanced iron metabolism triggered by IONPs appearance in blood stream after redistribution from the kidneys. Moreover, elevated Cu level in both the serum and kidneys after 7 days from the treatment may confirm the occurrence of IONPs reaccumulation within the renal pathways. On the other hand, increased Cu concentration on 7th day from IONPs injection may also indicate on IONPs induced long-term adverse effects e.g. inflammation 46,47. Thus, the elevated level of the element may be connected with systemic response to the oxidative stress which is accompanied by enhanced activity of Cu,Zn-SOD and/or ceruloplasmin. Increasing trend was observed for Cu concentration in the heart after 24 hours from subjection to the nanoparticles. The most possible reason for such anomaly is elevation of Cu level within the serum especially as the element contents both within the serum and the heart started to increase between 2nd and 24th hour from IONPs treatment. Ca is the microelement with versatile biological functions which controls multiple life processes 50–52. Ca has the largest gradient of any chemicals across the plasma membrane of all living cells: its extracellular concentration is 10 000-fold higher than the cytosol Ca content. Such disequilibrium is maintained by both the passive impermeability of the plasma membrane to Ca ions and by the active extrusion of Ca from the cell 53,54. Oxidative stress arising from e.g. exposure to oxidants like Fe2+ ions may simply alter Ca homeostasis leading to plasma membrane damage 52,53. Consequently, the large Ca gradient can no longer be maintained what results in excess Ca ions accumulation in the injured cell 53,54. The occurrence of these phenomena may explain both the elevated concentrations of Ca within the kidneys and upward trends of the element level in heart, which were observed after 2 hours and 7 days from IONPs injection. Zn, as an element responsible for proper folding and stability of Cu,Zn-SOD enzyme, is an important part of the antioxidant defense system against ROS 48,49. Cu,Zn-SOD, present in cytoplasm, nucleus and plasma of all living cells, catalyzes the superoxide anion radical (O2•-) dismutation into hydrogen peroxide (H2O2) 41,48,49,55,56. In case of ROS-induced oxidative stress the enzyme activity rises, thus re-establishing redox homeostasis within the affected tissue 52. The data presented in the Figure 5 revealed significantly increased Zn concentration within the kidneys and heart after 2 hours and 7 days from subjection to the NPs what may be explained by phenomenon of Cu,Zn-SOD activation resulting from IONPs-induced oxidative stress. What is more, the strong correlation between Zn and Ca levels as well as between the dynamics of their changes in animals treated with IONPs may further confirm the aforementioned explanation. Interestingly, the levels of Zn and Ca in the kidneys and heart seem to correlate also with Cu level in the serum. Furthermore, elevated concentration of Cu, probably connected with systemic releasing of ceruloplasmin being the positive acute phase protein, may additionally indicate at appearance of oxidative stress following inflammatory processes ongoing in the organism. It is also worth noticing, that although the NPs accumulation was not observed in the heart, the adverse effects of their administration and redistribution also affected this organ. Myocardium is one of the most sensitive tissues to Fe 57. It is believed that mechanisms of IONPs degradation in the body are very similar to those associated with ferritin in which the IONP core is dissolved in the acid environment of the lysosomes to free Fe ions 14,58. Thus the occurrence of the oxidative stress-mediated toxicity of the iron ions released from the IONP within heart tissue cannot be ruled out. However, to confirm such hypothesis, the assessment of the level of oxidative stress biomarkers such as lipid peroxidation level should be done 21,59,60. Hence challenge for future experiments is performing the biochemical analysis with use of Fourier transform infrared microspectroscopy, which will provide information on the content and structure of main biomolecules within the heart as well as the other organs 61. 11



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No statistically significant changes were observed within brain and spleen of rats subjected to NPs what may suggest the lack of PEG-IONPs influence on these organs. Such an observation is in consistency with the existing literature, suggesting that IONPs accumulation occurs within the spleen when their injected dose is 20 mg per 1 kg of body weight or higher 62, while the dose administered to the animals in current study was three orders of magnitude lower. In turn, crossing the blood-brain barrier by IONPs is possible only after applying of appropriate coating consisting of chitosan-PEG copolymer, wherein the hydrodynamic diameter of such particles cannot exceed 30 nm 63,64, which may explain the absence of the IONPs within the brain tissue. However, it is also possible that the elemental anomalies occurring within brain and/or spleen were limited to small tissue areas and might not be detected with the use of bulk TXRF analysis showing the averaged elemental changes within the organ. Therefore, the next step of the study should be the topographic elemental analysis using more sensitive and highly spatially resolved method such as X-ray fluorescence microscopy.

Conclusion Even low doses of intravenously injected IONPs may introduce significant elemental changes within kidneys and heart of treated animals. Although elevated Fe level, suggesting the presence of nanoparticles, was observed only within kidneys, the long-term elevation of Cu concentration accompanying enhanced iron metabolism was found not only in this organ but also within the heart and serum. What is more, increased levels of Cu, Zn and Ca in the kidneys and heart may indicate at progression of inflammation processes within the tissues. It may suggest that intravenously injected iron oxide nanoparticles could induce toxic effects within selected organs. Results obtained in frame of this paper allowed us to point out the target organs in which the NPs may accumulate as well as those in which elemental changes originating from IONPs systemic action can be observed. The presented data enabled us also to indicate the processes that may be responsible for the observed anomalies. Nevertheless, further research providing more detailed information about mechanisms underlying the presented adverse effects of subjection to the NPs and in particular relation between IONPs properties and systemic response to their intravenous administration are the challenging tasks for the future.

Supporting Information The median concentrations and the lowest detection limits (LLD) of Fe (Table S1), Cu (Table S2), Ca (Table S3) and Zn (Table S4) for the examined organs.

Acknowledgements Special thanks to PhD Eng. Przemyslaw Wachniew from Faculty of Physics and Applied Computer Science, University of Science and Technology in Krakow for providing access to chemical laboratory and sharing the mineralizer SpeedWave4. Special thanks to Prof. Piotr Warszynski and M.Sc. Aneta Kedra from The Jerzy Haber Institute of Catalysis and Surface Chemistry of the Polish Academy of Sciences in Krakow for DLS measurements with Nano ZS System. This work was partially financed by the Faculty of Physics and Applied Computer Science AGH UST statutory tasks as well as dean grant no 15.11.220.717/23 for PhD students and young researchers within subsidy of Polish Ministry of Science and Higher Education and the statutory research of the Institute of Zoology and Biomedical Research (Jagiellonian University) K/ZDS/007359. 12


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Disclosure Statement The authors report no conflicts of interest.

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Low Doses of Polyethylene Glycol Coated Iron Oxide Nanoparticles

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