Superparamagnetic Iron Oxide Nanoparticles as Novel X-ray

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Superparamagnetic Iron Oxide Nanoparticles as Novel X-ray Enhancer for Low-dose Radiation Therapy Stefanie Klein, Anja Sommer, Luitpold Distel, Jean-Louis Hazemann, Wolfgang Kroener, Winfried Neuhuber, Paul Muller, Olivier Proux, and Carola Kryschi J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 15 May 2014 Downloaded from http://pubs.acs.org on May 16, 2014

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The Journal of Physical Chemistry

Superparamagnetic Iron Oxide Nanoparticles as Novel X-Ray Enhancer for Low-Dose Radiation Therapy Stefanie Klein,a Anja Sommera, Luitpold V. R. Distelb, Jean-Louis Hazemannc, Wolfgang Krönerd, Winfried Neuhubere, Paul Müllerd, Olivier Prouxf, Carola Kryschi*a a

Department Chemistry and Pharmacy, Physical Chemistry I and ICMM, Friedrich-Alexander

University Erlangen, Egerlandstr.3, D-91058 Erlangen, Germany. b

Department of Radiation Oncology, Friedrich-Alexander University Erlangen,

Universitätsstr. 27, D-91054 Erlangen, Germany. c

Institut Néel, UPR 2940, CNRS-Université Joseph-Fourier, 25 avenue des Martyrs, BP 166,

F-38042 Grenoble cedex 9, France. d

Department of Physics, Experimental Physics, Friedrich-Alexander University Erlangen,

Erwin-Rommel-Str. 1, D-91058 Erlangen, Germany. e

Department of Anatomy, Chair of Anatomy I, Friedrich-Alexander University Erlangen,

Krankenhausstr. 9, D-91054 Erlangen, Germany. f

Observatoire de Sciences de l`Univers de Grenoble, UMS 832, CNRS-Université Joseph-

Fourier, F-38041 Grenoble cedex 9, France.

AUTHOR INFORMATION Corresponding Author *Prof. Dr. Carola Kryschi Dept. Chemistry and Pharmacy Friedrich-Alexander University Erlangen Egerlandstr. 3 D-91058 Erlangen, Germany Phone: +49-9131-8527307 e-mail: [email protected]

Notes The authors declare no competing financial interest.

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ABSTRACT Superparamagnetic iron oxide nanoparticles (SPION) with a mixed phase composition (γFe2O3)1–x(Fe3O4)x and sizes between 9 and 20 nm were synthesized via co-precipitation and were either left uncoated or subsequently surface-stabilized with citrate or malate anions. The sizes, morphology, surface chemistry and magnetic properties of the nanoparticles were characterized using transmission electron microscopy (TEM), FTIR spectroscopy and superconducting quantum interference device measurements, respectively. Cellular uptake and intracellular distribution in normal tissue and tumor cells were verified by TEM images. X-ray induced changes of the oxidation state and site geometries of surface iron ions of uncoated and citrate-coated SPION were explored by collecting Fe K-edge X-ray absorption spectroscopy data. The potential applicability of citrate- and malate-coated SPION as X-ray enhancer for radiation cancer therapy was substantiated by their drastic enhancement of the concentration of reactive oxygen species (ROS) in X-ray irradiated tumor cells.

Keywords: superparamagnetism, X-ray treatment, MCF-7 cells, Caco-2 cells, reactive oxygen species, Fenton raction


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INTRODUCTION

In radiation cancer therapy tumor tissue is mainly destroyed due to the interaction of highenergy ionization radiation with the cytosol and nucleus of tumor cells in the irradiated volume. The cellular response consists in DNA double- and single-strand breaks as well as in the excitation and ionization of water, oxygen and other molecules in the cytosol. To minimize undesirable side effects for normal tissue cells in the irradiated volume, low linear energy transfer radiation as being X-rays and γ-rays is nowadays used in radiotherapy. The cytotoxicity of X-rays predominantly arises from the generation of free radicals in the cytosol. These reactive oxygen species (ROS) cause DNA double-strand breaks in the nucleus and thereupon, preferentially induce apoptosis of cancer cells as exhibiting a high proliferation rate and a reduced ability of DNA self-repair.1,2,3 In contrast to chemotherapeutics, X-rays can be locally administered to malignant cells by focusing onto tumor tissue, where the X-rays penetrate cellular boundaries and develop anticancer effects by generating ROS in the cytosol.

However, the curative potential of X-ray treatment for cancer therapy is often limited by its toxic effect on normal tissue and the intrinsic radioresistance of tumor cells. The tumor radiosensitivity is regulated by the balance between X-ray induced DNA damage and distinct DNA repair processes.4 Since H2O is the most abundant intracellular molecule, the exposure of cells to X-rays gives rise to decomposition reactions of water. In normoxic cells X-rays generate a variety of ROS that are highly reactive radicals with essential functions in living organisms.5 A moderate increase of the ROS concentration can promote cell proliferation and differentiation,6,7 whereas excessive ROS concentrations cause oxidative damage to lipids, proteins and DNA.8 In particular, DNA double-strand breaks may activate a complex machinery of DNA damage recognition, repair and response processes.4 Unfortunately the exact mechanism is only rudimentarily understood. One major and hardly conquerable obstacle for any cancer therapy is the hypovascularity of most solid tumors. These tumors microscopically comprise heterogeneous hypoxic regions due to limited diffusion of oxygen throughout their tissue. Since the ROS generation requires a sufficiently high oxygen level, any kind of radiotherapy is less effective for hypoxic cancer cells.9

To overcome radioresistance and therewith, to enhance the effectiveness of ionizing radiation, chemical and nanoparticulate radiosensitizers have been developed which specifically increase the sensitivity of tumor cells to X-rays. Nowadays, there is a broad spectrum of



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radiosensitizers that differ in their chemical composition, physical, pharmacological properties and intracellular functionality when interacting with X-rays. Gold nanoparticles, for instance, may increase the primary radiation damage via their emission of Compton electrons, photoelectrons or Auger electrons,10 whereas more specifically acting molecular agents can inhibit the DNA damage repair,11 change the cell cycle and thus, enhance the radiosensitivity by minimizing the X-ray induced Gap2/Mitosis (G2/M) arrest,12 or promote the apoptosis.13 In comparison with normal tissue cells tumor cells often contain an increased ROS concentration. Therefore the lethal effect of X-rays on tumor cells is considerably larger than that for normal tissue cells.14,15

Superparamagnetic iron oxide nanoparticles (SPION) were reported to substantially enhance the impact of X-rays in tumor cells by catalyzing the ROS formation.16 SPION usually consist of a superparamagnetic single crystalline core with magnetite (Fe3O4), maghemite (-Fe2O3) composition or a mixed phase composition (γ-Fe2O3)1–x(Fe3O4)x with x increasing with the nanoparticle size.17 Magnetite as well as maghemite crystallizes in an inverse spinel structure with Fe3+ ions on tetrahedral and octahedral lattice sites, whereas the Fe2+ ions of magnetite are randomly distributed in octahedral sites. Surface structures of uncoated SPION in aqueous media predominantly consist of amphoteric hydroxyl groups that are electrostatically bound to surficial Fe2+ and Fe3+ ions. Coating of SPION with surfactants such as citric acid and other carboxylic acids may provide surface stabilization and support cellular uptake.16,18,19 The superparamagnetic behavior of SPION, as required for their desired magnetic field-guided transport to the tumor tissue, crucially depends on both, core size and surface coating. The limiting size of SPION for in vivo applications is 40 nm.20 In general, superparamagnetism scales with the nanoparticle size and may be reduced by bulky surface structures. Therefore, ideal surface coatings are those with thicknesses smaller than 1 nm, since such thin coatings hardly impair the magnetization of the SPION in the external inhomogeneous magnetic field. Furthermore, the chemical composition and charges of surface structures rule the pathway of cellular uptake and control the intracellular distribution as well as may provide biocompatibility for internalized SPION. While the latter is best achieved by biopolymer or biomolecular coatings, uncoated SPION exhibit undesired cytotoxicity.20,21,22 Degradation and metabolism of uncoated SPION occur via the release of Fe3+ and Fe2+ ions into the cytoplasm, where the iron ions were reported to adversely affect intracellular oxido-reduction reactions and homeostasis of ROS.21 In addition, intracellular iron ions may initiate the Fenton reaction and Haber-Weiss cycle and thereupon, enhance the ROS formation.23,24 Voinov et al. could


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substantiate by in vitro experiments that SPION surfaces are more efficient catalysts for the ROS formation than free iron ions.21 In a recent study, we could show the applicability of SPION as X-ray sensitizer for breast tumor cells.16 The radiosensitizing effect especially persists in the X-ray induced activation of the SPION surfaces for catalyzing the ROS generation through both, Haber-Weiss cycle and Fenton reaction.

In this contribution we examined X-ray induced changes of the surface structures of surfacemodified SPION and their influence on the ROS formation in tumor and normal tissue cells. Therefor human colon cancer cells (Caco-2), human breast cancer cells (MCF-7) and mouse fibroblast (3T3) cells were incubated with uncoated, citrate- and malate-coated SPION and exposed to X-rays at single doses of 1 and 3 Gy, respectively. The size distributions and shapes of the SPION were studied using transmission electron microscopy (TEM). X-ray induced alterations of the surface chemistry of uncoated and citrate-coated SPION were elucidated employing X-ray absorption spectroscopy. The cellular uptake and intracellular distribution of uncoated and coated SPION in normal and tumor cells were visualized using TEM. The X-ray enhancing effect of coated SPION in tumor cells was unambiguously verified via the detection of significantly increased ROS concentrations.

EXPERIMENTAL SECTION

Chemicals FeCl3×6H2O (99%, Acros organics), FeCl2×4H2O (99%, Sigma Aldrich), citric acid anhydrous (99.5%, Alfa Aesar) and DL-malic acid (99%, Fluka) were used as received. Dulbecco’s modified eagle medium (DMEM), L-glutamine, fetal calf serum (FCS), penicillinstreptomycin-solution, sodium pyruvate, phosphate buffered saline (PBS), non-essential amino acids (MEM), trypsin/EDTA, 3-(4,5-dim-ethythiazol-2-il)-2,5-diophenyltetrazolium bromide) MTT (98%), 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (95%) were purchased from Sigma-Aldrich and glutaraldehyde (25 %) from Roth. DCFH-DA was dissolved in dimethyl sulfoxide (DMSO) (99.7 %, Baker) to obtain a stock solution (0.01 M) and was kept frozen at -20°C. For loading the cells with DCFH-DA the stock solution was mixed with DMEM to a final concentration of 100 µM.



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Synthesis of iron oxide nanoparticles SPION syntheses were carried out by co-precipitation of ferric and ferrous chloride at 0°C under oxygen-free conditions following Massart’s method.25 SPION were left uncoated or subsequently surface stabilized with citric acid or malic acid so that citrate- and malate-coated SPION were obtained.

Characterization FTIR spectra of uncoated, citrate- and malate-coated SPION were recorded on a FTIR spectrometer (Prestige-21 Shimadzu) using KBr pellets. The magnetization of the SPION powder samples were measured using a Quantum Design MPMS-XL5 SQUID magnetometer. The respective value of saturation magnetization was determined under an applied constant magnetic field of 5 T at 300 K. TEM images of the SPION were taken using a Zeiss EM 900 instrument operating at 80 kV accelerating voltage. SPION loaded 3T3 and MCF-7 cells were imaged using a Zeiss 906 transmission electron microscope (LEO, Oberkochen, Germany). The concentrations of the SPION sample solutions were determined using ICP-AES (Perkin Elmer Plasma 400). The MTT assay was measured at 550 nm employing an Elisa microplate reader (Dynatech Laboratories, Inc.). The different cells experiments were irradiated using a 120 kV X-ray tube (Isovolt Titan, General Electrics, Ahrensberg, Germany).

Fe K-edge X-ray absorption spectroscopy experiments Fe K-edge X-ray absorption spectroscopy (XAS) experiments were performed on the FAME beamline at the European Synchrotron Radiation Facility in Grenoble (ESRF).26 X-ray absorption near edge structure spectra (XANES) and extended X-ray absorption fine structure (EXAFS) spectra were recorded at liquid-helium temperature (10 K) in the fluorescence mode using Si (220) monochromator crystals. The energy was calibrated by measuring the XANES of a Fe metal foil (first maximum of the derivative set at 7112 eV). The XAS samples were prepared as pellets of homogeneous fine powders of SPION diluted in boron nitride.

Cell culture Human breast cancer cells (MCF-7), human colon cancer cells (Caco-2) and mouse fibroblasts (3T3) were cultured in DMEM containing 4500 mg glucose/L, which was enriched with 10 % fetal calf serum (FCS), 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine and 1% MEM nonessential amino acids. In a humidified environment of 5 % CO2 at 37 °C the cells were incubated and sub-cultivated twice a week.


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Transmission electron microscopy (TEM) The different cell lines were incubated with cell culture medium containing uncoated or coated SPIONs at a concentration of 0.1 mg Fe/mL. Cells were washed with PBS and fixed with 2.5 % glutaraldehyde overnight at 4°C and then post-fixed in 1% osmium tetroxide and 3 % potassium ferricyanide at room temperature. Through graded alcohols cells were dehydrated, embedded in Epon and mounted on Epon blocks. Un-contrasted silver-grey ultrathin sections were imaged.

Cell viability assay Mitochondrial function and cell viability of the SPION were assessed using the 3-(4,5dimethylthiazol)-2-diphenyltetrazolium bromide (MTT) assay. The different cell lines were seeded in a 96 well-plate at a density of 103 cells per well. After 3 days the cell culture medium was replaced with one containing uncoated or coated SPION at a concentration of 0.1 mg Fe/mL. After 24 h incubation 50 µL of MTT solution (0.5 mg/mL in PBS) was added. The solution was carefully removed after 1 h and the formazan crystals were solubilized with 100 µL DMSO. The metabolic activity was determined by measuring the absorbance of the formazan solution at 590 nm.

Intracellular ROS measurement MCF-7, Caco-2 or 3T3 cells were cultivated in 96 well-plates at a density of 103 cells per well and were allowed to grow over 3 days. After removing the medium, the cells were incubated for 24 h with cell culture media that contains SPION (0.1 mg Fe/mL). Afterwards the cells were washed with PBS and loaded with 100 µM DCFH-DA in DMEM for 30 min. Each well was loaded with PBS. One half of the plate was irradiated at a single dose of 1 Gy or 3 Gy. Intracellular DCFH was quantitatively oxidized by ROS to the fluorescent DCF dye. Therefore DCF fluorescence intensity is directly proportional to the ROS concentration. The fluorescence emission was excited at 480 nm, and its spectrum was recorded in the range of 500 - 700 nm. Values of the relative fluorescence intensity were obtained by integrating the spectra. The obtained values of fluorescence intensities were related to those obtained from fluorescence measurements of cells in culture medium



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Statistical analysis Data are presented as arithmetic mean values ± standard error (SE). Statistical analysis was performed using the analysis of variance (ANOVA) with post hoc Bonferroni correction for multiple comparisons. A value of p < 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

The focus of this contribution is set on the examination and verification of applicability of SPION as radiosensitizer in tumor cells for X-rays at clinical doses. Therefor Caco-2, MCF-7 and 3T3 cells were incubated with uncoated, citrate-coated or malate-coated SPION and exposed to X-rays at single doses of 1 Gy or 3 Gy. Uncoated and coated SPION possess quasi-spherical shapes and exhibit size distributions between 9 and 20 nm for uncoated SPION, between 7 and 17 nm for citrate-coated and 6 and 16 nm for malate-coated SPION as being exemplarily visualized by the TEM image of malate-coated SPION in Figure 1. The magnetic properties of uncoated, citrate- and malate-coated SPION were investigated by measuring their magnetization at magnetic field strengths between -5 and 5 T. Figure 2 depicts the magnetization measurements performed on all three powder samples at 300 K. The hysteresis loops of the magnetization measurements do not show any remanence or coercivity which indicates perfect superparamagnetism for all SPION samples. The value of saturation magnetization (MS) determined for uncoated SPION is about 77 emu/g. The slightly decreased MS values of malate-coated and citrate-coated SPION, with 74 and 70 emu/g, respectively, may be explained with a weak shielding effect of the respective coating layer. The surface chemistry of the SPION was examined using FTIR transmission spectroscopy (Figure 3). The FTIR spectra of the uncoated (black), citrate-coated (red) and malate-coated (blue) SPION comprise all a strong transmission band around 580 cm-1 which is attributed to the (Fe-O) stretching vibration. The spectrum of the uncoated SPION contains a vibrational band at 3400 cm-1 that is assigned to the (O-H) stretch and a smaller band at 1630 cm-1 due to the (O-H) deformation vibration. The spectra of citrate- and malate-coated SPIONs exhibit in addition two prominent vibrational peaks around 1398 and 1630 cm-1 which are attributed to the (O-H) deformation and (C=O) stretching vibration, respectively. Both vibrational bands identify the citrate and malate moieties and their covalent bonding to the SPION surface.


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Complementary information of the oxidation states and local site geometries of surface iron ions of non-irradiated and X-ray irradiated SPION was obtained from Fe K-edge XANES and EXAFS. Figure 4 presents the normalized Fe K-edge XANES of uncoated and citrate-coated SPION powder samples that were either exposed to X-rays at a single dose of 3 Gy (red and and blue line) or left non-irradiated (green and black line). For comparison, the normalized XANES of bulk maghemite (magenta line) and magnetite (orange line) are depicted in Figure 41(a). The pre-edge peak of the XANES spectra (7114 eV) is attributed to quadrupole transitions from 1s to 3d orbitals with 3d-4p mixing, while the prominent absorption edge (i.e. the white-line at 7133 eV) and higher-energy features arise from dipole-allowed 1s to 4p continuum-states transitions and multiple scattering due to the Fe environment.28,29,30,31 Fe3O4 and -Fe2O3 exhibit XANES that differ in both, the overall spectral shape and the absorptionedge energy.28,29 This is due to the presence of Fe2+ ions in Fe3O4 and the crucial dependence of the absorption-edge energy on the Fe-O bond length. Since the mean interatomic distance in -Fe2O3 is shorter than that for Fe3O4, the absorption edge peak of the -Fe2O3 XANES is shifted for ca. 1.5 eV to higher energy values and is more pronounced in comparison with the Fe3O4 XANES (Figure 4(a)). We measured the Fe K-edge XANES in the fluorescence mode, in order to increase the sensitivity of this spectroscopy technique to the Fe2+:Fe3+ ratio and site geometries of surface iron ions. For uncoated SPION the Fe2+ and Fe3+ ions are presumably terminated with electrostatically bound hydroxyl groups and physisorbed water molecules. A more effective protection against surficial redox reactions and ligand exchange is achieved through bidentately or tridentately coordinating citrate ligands.18 This hypothesis is manifested by the differences in the respective XANES: the spectrum of the citrate-coated SPION (green solid line) is slightly shifted to lower energies. The absorption-edge peak is less pronounced when compared to the spectrum of uncoated SPION (black solid line). This implies that the citrate-coated SPION surface composition exhibits a relatively higher Fe2+:Fe3+ ratio. After irradiation with X-rays the surfaces of both, uncoated and citrate-coated SPION, will be completely freed from any chemisorbed or electrostatically bound molecule. In comparison with the XANES of the uncoated SPION (black solid line) the XANES of Xray irradiated, uncoated SPION (red solid line) is slightly red-shifted and contains a relatively lowered absorption edge peak. These spectral differences (Figure 4(b), magenta solid line) clearly indicate an increase of surface Fe2+ ions which might arise from X-ray induced ablation of Fe3+ ions. For citrate-coated SPION the impact of X-rays on the surface Fe2+/Fe3+ composition is converse as visualized by the difference spectrum in Figure 4(c) (magenta solid line). The XANES of the X-ray exposed citrate-coated SPION displays the overall



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spectral shape of that of non-irradiated uncoated SPION (Figure 4(a)). It is here suggested that the ablation of the outer surface layer is associated with an alteration of the chemical composition of the surface. Presumably, X-ray induced removal of the citrate ligands provokes the oxidation of Fe2+ to Fe3+. In a nutshell, the XANES results show that the interaction of uncoated and citrate-coated SPION with X-rays led to the creation of novel surface structures and composition which provided highly reactive surfaces. It has to be mentioned that the XANES SPION samples under study were examined 3 days after their Xray treatment. Thus the Fe2+:Fe3+ ratio of the surface composition might have changed by aging that crucially depends on the environment of the SPION. This implies that SPIONinternalized by cells experience another composition of solvation shells than dry SPION samples.

The intracellular distribution of uncoated and citrate- and malate-coated SPION in normal and tumor cells were examined using TEM. Figure 5 shows TEM images of MCF-7 (A) and 3T3 (B) cells, both were incubated with either uncoated or citrate-coated SPION. Caco-2 cells (C) were loaded with uncoated SPION. While the incubation with uncoated SPION in MCF-7 cells took 24 h, 48 h incubation was required for a sufficient cellular uptake by 3T3 cells. Since the uncoated SPION were observed to predominantly sojourn in vesicles into the cytoplasm (A1, B1, C1), their internalization probably had occurred via endocytosis. This is indicated by the pseudopodia, which were built from the cell membrane towards the SPION (C1). In the Caco-2 cells more of uncoated SPION were released into the cytoplasm (C2) than in the MCF-7 and 3T3 cells. On the other hand, the TEM images of the cells incubated with citrate- or malate-coated SPION show a completely different scenario. Citrate-acid coated SPION formed large agglomerates in the cytoplasm of the MCF-7 cells (A2), whereas inside 3T3 cells (B2) the SPION appeared to be lined up along the endoplasmic reticulum. The citrate and malate ligands presumably facilitated the membrane crossing of the SPION and thereupon, promoted faster cellular uptake and efficient release from the subsequently formed vesicles. Another result of this study is the observation that the MCF-7 and Caco-2 cells internalized the SPION much faster and at a higher concentration than the normal tissue 3T3 cells were doing. This is due to the higher metabolism of tumor cells.

The biocompatibility of the uncoated, malate- or citrate-coated SPION was assessed using the MTT assay. Figure 6.A illustrates the relative cell viability (%) of the Caco-2, MCF-7 and 3T3 cells which were incubated with coated and uncoated SPION for 24 h. The MCF-7 and


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3T3 cells exhibit very similar and relatively high values of cell viability for all SPION samples. Uncoated SPION turned out to be a little toxic for Caco-2 cells (70%), whereas coated SPION exhibit a higher biocompatibility for Caco-2 cells than for MCF-7 and 3T3 cells. Caco-2 cells show, when containing malate-coated SPION, a survival rate of 94 % and with internalized citrate-coated SPIONS ideal cell viability (100 %). Uncoated and coated SPION differ in their surface chemistry and therefore lead to a distinct chemical response of the cytoplasm. For instance, uncoated or partially coated SPION may react in the cytoplasm via their surface iron ions with hydrogen peroxide and thereupon catalyze the formation of the cytotoxic hydroxyl radical as being a ROS. The influence of coated and uncoated SPION on the ROS formation in 3T3, MCF-7 and Caco-2 cells was studied by measuring the relative fluorescence intensity of the DCF dye which scales with the ROS concentration (Figure 6.B). After 24 h incubation with coated SPION the ROS level in 3T3, Caco-2 and MCF-7 cells is only slightly increased which nicely agrees with the results obtained from the cell viability assay. Uncoated SPION in 3T3 and MCF-7 cells also show a negligibly enhancing effect on the ROS formation. On the other hand, uncoated SPION internalized in Caco-2 cells raised the ROS concentration for 60 %, which is consistent with the verification of the cytotoxicity assessed by the MTT assay. Uncoated SPION were trapped in vesicles in MCF-7 and 3T3 cells, where they were not able to catalyze the Fenton or Haber-Weiss reaction. In contrast, a higher concentration of the uncoated SPION was observed in the cytoplasm in Caco-2 cells, which explains the higher ROS production due to the catalytic activity of the SPION surfaces or the release of iron ions. The impact of X-rays on surface structures and composition of uncoated and coated SPION and their altered reactivity in the cytoplasm was examined by measuring the relative ROS concentration of X-ray exposed normal and tumor cells (Figure 7). Caco-2 cells incubated with uncoated, citrate- or malate-coated SPION were irradiated with X-rays at a single dose of 1 Gy (Figure 7.A). In comparison with non-irradiated Caco-2 cells, citrate-coated and malate-coated SPION increased the ROS concentration for 388 % and 369 %, respectively, whereas uncoated SPION did not show any effect on the ROS formation under X-ray exposure. Similarly, the MCF-7 cells developed a higher ROS formation for coated SPION in comparison to uncoated SPION: citrate and malate-coated SPION caused a rise of the ROS level for 327 % and 294 %, respectively, whereas the increase of the ROS level is about 10% for uncoated SPION. This large difference in the ability of uncoated and coated SPION for the ROS formation in tumor cells arises from the distinct surface composition and structures which provide the interaction with X-radiation. Uncoated SPION internalized in non-irradiated Caco-2 cells show cytotoxicity because of their enhancement of



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ROS formation, which might be understood to arise from unsaturated Fe2+ ions in their surfaces. X-radiation of intracellular uncoated SPION only hardly increased the content of reactive surficial Fe2+ ions. In contrast, citrate- and malate-coated SPION did not exhibit any cytotoxicity in non-irradiated tumor cells. Intracellular coated SPION, when exposed to Xradiation, obtained highly reactive, catalytically acting surfaces that drastically increased the ROS formation in tumor cells. On the other hand, neither uncoated nor coated SPION increase the ROS formation in the normal tissue 3T3 cells. These experimental results establish a quite promising foundation for their future application in cancer therapy. Another future-indicating result is the observation that a single X-radiation dose of 3 Gy did not further boost the ROS formation in 3T3, MCF-7 and Caco-2 cells, independently of containing uncoated or coated SPION (Figures 7.B and C). Analogous experiments with Caco-2 cells were performed using citrate-coated and malate-coated SPION that were stored under ambient conditions for different periods (Figure 8). Rising the storage time of the coated SPION from 1 to 3 weeks, the relative ROS concentration was measured to be lowered to 150 % for the malate coating and to 70 % in case of citrate ligands. Apparently, the SPION surfaces undergo an aging process that takes place as oxidation of Fe2+ to Fe3+ ions. Since Fe2+ ions catalyze via the Fenton reaction the formation of the hydroxyl radical, stored SPION had partially lost their catalytic activity and thus, increased the ROS formation with a considerably reduced extent.

CONCLUSION

To engineer potent X-ray enhancer for the radiation cancer therapy, uncoated and citrate- and malate-coated SPION with a mixed phase composition (γ-Fe2O3)1–x(Fe3O4)x and sizes between 9 and 20 nm were synthesized and characterized using TEM, XANES and EXAFS, FTIR spectroscopy, SQUID and cell viability tests. The superparamagetism of the SPION was confirmed and values of the saturation magnetism between 70 and 77 emu/g were measured. The citrate and malate ligands were found to be covalently bound at the SPION surfaces. The impact of X-rays on uncoated and coated SPION resulted into the creation of novel surface structures and composition which provided highly reactive surfaces in case of citrateterminated SPION. The cellular uptake of the citrate- and malate-coated SPION by MCF-7 and Caco-2 cells was observed to occur very efficiently via endocytosis. In contrast to uncoated SPION, the coated ones are biocompatible. As shown by measuring the relative DCF fluorescence intensity the normal tissue and tumor cells, the interaction between X-rays


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at a dosage of 1 Gy and intracellular coated SPION led to a drastic increase of the ROS concentration for more than 300 %, while intracellular uncoated SPION did not affect the ROS production of the cells under study. In addition, neither uncoated nor coated SPION increase the ROS formation in normal tissue 3T3 cells. Furthermore, in comparison with the X-ray treatment at 1 Gy a higher X-ray dosage of 3 Gy was shown to slightly decrease the ROS production in tumor cells. All these results show that citrate- and malate SPION act as X-ray enhancer by preferentially catalyzing the ROS formation in tumor cells. As being nontoxic these SPION are best suited for the application as radiosensitizer in cancer radiation therapy.

ACKNOWLEDGEMENTS Support of the Deutsche Forschungsgemeinschaft (graduate school 1161/2) is gratefully acknowledged. We thank Andrea Hilpert for TEM imaging studies (Institute of Anatomy I, Friedrich-Alexander University Erlangen-Nuremberg). Sincere thanks are given to PD Dr. Oliver Zolk (Institute of Experimental and Clinical Pharmacology and Toxicology, FriedrichAlexander University Erlangen-Nuremberg) for generously supporting our cell viability assay experiments. Furthermore, we are grateful to Andreas Postatny for conducting so diligently the ICP-AES experiments on our samples. (Prof. Dr. Peter Wasserscheid, Institute of Chemical Reaction Technique, Friedrich-Alexander University Erlangen-Nuremberg).

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Figure Captions

Fig.1. TEM image of malate-coated SPION. Fig.2. Magnetization curves of uncoated SPION (black solid line), citrate-coated SPION (blue solid line) and malate-coated SPION (red solid line) at 300 K. Fig.3. FTIR transmission spectra of uncoated SPION (black solid line), citrate-coated SPION (red solid line) and malate-coated SPION (blue solid line). Fig. 4. (a): Normalized Fe K-edge XANES of uncoated SPION (black solid line) and citratecoated SPION (green solid line), X-ray irradiated SPION (red solid line), X-ray irradiated citrate-coated SPION (blue solid line), bulk maghemite (orange solid line) and bulk magnetite (magenta solid line); (b): XANES of X-ray irradiated SPION (red solid line) and SPION (black solid line) and their difference (magenta solid line); (c): XANES of X-ray irradiated citrate-coated SPION (blue solid line) and citrate-coated SPION (green solid line) and their difference (magenta solid line). Fig. 5 TEM images of MCF-7 (A), 3T3 (B) and Caco-2 (C) cells: after 24 h incubation uncoated SPION were incorporated in vesicles in MCF-7 cells (A1) and after 48h incubation in vesicles in 3T3 cells (B1). Uncoated SPIONs were uptaken via endocytosis in Caco-2 cells (C1) and afterwards released into the cytoplasm (C2). Citrate-coated SPION agglomerated in the cytoplasm of MCF-7 cells (A2) and were adsorbed along the endoplasmic reticulum in 3T3 cells (B2). Fig. 6 Biocompatibility of the uncoated, citrate- and malate-coated SPION were tested using the MTT assay and ROS assay: (A) the metabolic activity (%) of Caco-2 (black), MCF-7 (grey) and 3T3 (white) cells, n = 6, *p > 0.05; (B) the relative DCF fluorescence intensity as a measure for the ROS concentration in the non-irradiated cells, n = 4, *p > 0.05. Fig. 7 Relative DCF fluorescence intensity as a measure of the ROS concentration in X-Ray irradiated and non-irradiated Caco-2, MCF-7 and 3T3 cells loaded with citrate-, malate-coated or uncoated SPION: (A) irradiation of the Caco-2 MCF-7 and 3T3 cells with 1 Gy, X-ray treated, SPION loaded Caco-2 cells (B) and (C) 3T3 cells at dosages of 1 and 3 Gy; all values are represented as increase of relative DCF fluorescence intensity (%), n = 4, *p > 0.05. Fig. 8 Relative DCF fluorescence intensity as a measure of the ROS concentration in X-Ray irradiated and non-irradiated Caco-2, MCF-7 and 3T3 cells loaded with citrate-,



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malate-coated or uncoated SPION which were stored 1 week, 2 weeks and 3 weeks, n = 4, *p > 0.05.


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Figures

Figure 1

80

SPION MA-SPION CA-SPION

60

M [emu/g]

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40 20 0 -20 -40 -60 -80 -6

-4

-2

0

2

µ0H [T]

Figure 2 ACS Paragon Plus Environment

4

6


uncoated SPION citrate-coated SPION malate-coated SPION

80 70

transmission

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60 50 40 30 20 10 0 4000

3000

2000

1000

wavenumber [cm-1]

Figure 3


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Intensity [a.u.]

(a)

bulk maghemite bulk magnetite X-ray treated SPION@CA SPION@CA X-ray treated SPION SPION

7100

7120

7140

7160

7180

Energy [eV]

(b)

Intensity [a.u.]

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X-ray treated SPION SPION difference

7100

7120

7140

7160

7180

Energy [eV]


7200

7200


(c)

Intensity [a.u.]

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X-ray treated SPION@CA SPION@CA difference

7100

7120

7140

7160

7180

Energy [eV]

Figure 4


7200

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Figure 5



A

Cell Viability (% of control)

150

*

100

*

** 50

m al

N

ci tr at

at eco at e

eco at ed

d

SP I

SP IO

O N

SP IO N

0

B

200

Caco-2 MCF-7 3T3

**** 150 100 50

SP IO

O

N

N

N

0

oa te at ec ci tr

m

al at e

-c o

at ed

d

SP I

SP IO

% Increase of Fluorescence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 6


Caco-2 MCF-7 3T3

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A

800

SPION malate-coated SPION citrate-coated SPION

***

600

** ***

**** 400 200 0

Caco-2

MCF-7

3T3

B

800 600

1 Gy 3 Gy

***

***

**

**

400 200

N

ci tr at eco at ed

SP IO al at eco at ed m

SP IO

N

N

0

SP IO


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




150

1 Gy 3 Gy

100

50

N

ci tr at eco at ed

m

al at eco at ed

SP IO

SP IO

N

N

0 SP IO


C

Figure 7


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800 600

SPION malate-coated SPION citrate-coated SPION

**** ****

400

*

*

200 0

1. week 2. week 3. week

Figure 8


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TOC Graphic

uncoated SPION

coated SPION

1 Gy

nucleus

nucleus

1 Gy

H 2O 2 OH

.


Superparamagnetic Iron Oxide Nanoparticles as Novel X-ray

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