A Facile One-Pot Synthesis of Water-Soluble , Patchy Fe 3 O 4-Au Nanoparticles for Application in Radiation Therapy

A facile one-pot synthesis route for the preparation of water-soluble, biocompatible patchy Fe3O4-Au nanoparticles (Fe3O4-Au pNPs) was developed. Biocompatibility was attained through surface functionalization with 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide. The morphology, composition, crystal structure and magnetic properties of the Fe3O4-Au pNPs were investigated by conducting experiments with transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction and superconducting quantum interference device, respectively. Internalization of the Fe3O4-Au pNPs by MCF-7 cells occurred via endocytosis. The performance of the Fe3O4-Au pNPs as X-ray radiosensitizer in tumor cells was compared with that of gold nanocluster and Fe3O4 NPs. For this reason, MCF-7, A549 and MCF-10A cells were loaded with the respective kind of nanoparticles and treated with X-rays at doses of 1, 2 or 3 Gy. The nanoparticle-induced changes of the concentration of the reactive oxygen species (ROS) were detected using specific assays, and the cell survival under X-ray exposure was assessed employing the clonogenic assay. In comparison with the gold nanocluster and Fe3O4 NPs, the Fe3O4-Au pNPs exhibited the highest catalytic capacity for ROS generation in MCF-7 and A549 cells, whereas in the X-ray-induced ROS formation in healthy MCF-10A cells was hardly enhanced by the Fe3O4 NPs and Fe3O4-Au pNPs. Moreover, the excellent performance of Fe3O4-Au pNPs as X-ray radiosensitizers was verified by the quickly decaying radiation dose survival curve of the nanoparticle-loaded MCF-7 and A549 cells and corroborated by the small values of the associated dose-modifying factors.


Introduction
Over the last century, high-energy radiation, and in particular X-rays, has been utilized to treat cancer.The underlying concept is that rapidly proliferating cancer cells are more sensitive to X-rays than healthy cells [1].X-ray irradiation of the cytoplasm causes radiolysis of water and gives rise to the formation of reactive oxygen species (ROS), including the super oxide radical (O 2 •-), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (HO • ).The most reactive one, HO • , provokes severe cellular damage, as it interacts with all biological molecules.This may give rise to lipid peroxidation, protein damage, deoxyribonucleic-acid strand breakages and membrane destruction which results in apoptotic and necrotic cell death.Despite substantial advances in the progress of novel cancer therapies such as the successful development of brachytherapy and intensity-modulated radiotherapy, nowadays, the greatest challenge is still to expose tumor cells to a lethal dose of X-rays while sparing normal cells [2].An encouraging approach is the application of nanoparticulate radiosensitizers to tumor tissue which boosts the impact of X-rays on tumor cells [3][4][5][6][7][8][9].Local dose enhancement in radiotherapy of cancer employs high-Z materials (e.g., gold, platinum, bismuth and gadolinium), as they exhibit a large photoelectric absorption coefficient [5].Interaction between X-rays with moderate energies (10 to 500 keV) and high-Z gold atoms in nanoparticles generates secondary electrons, such as photoelectrons and Auger electrons [4,8].Since the range of these electrons is very short, they are highly effective in close proximity to gold nanoparticles inside the tumor cell.X-radiation-induced emission of these short-range electrons facilitates the deposition of highly localized energy into the cellular organelles, which inflicts irreversible damage to the targeted cells.On the other hand, due to their rapid decay within nanoscale volumes, these electrons cannot impair the surrounding healthy tissue.Therefore, gold nanoparticles (AuNPs) are auspicious candidates for application as radio-enhancers for X-rays in radiotherapy [6,[10][11][12].
The most effective reactive oxygen species (ROS), which are generated by X-ray irradiation of intracellular AuNPs, are the superoxide anion (O 2 •-) and hydroxyl radical (HO • ).X-ray-triggered generation of photoelectrons and Auger electrons is understood to be responsible for the formation of O 2 •-in the vicinity of gold nanoparticles, whereas the emitted characteristic X-radiation causes the formation of HO • due to water radiolysis [13].Among these ROS, the HO • causes the greatest damage to tumor cells by oxidizing lipids, proteins and DNA [14].In recent studies [15,16], we could unambiguously prove that intracellular Fe 3 O 4 nanoparticles (SPIONs) enhanced the impact of X-radiation on tumor cells by significantly increasing the ROS production and, in particular, by raising the intracellular HO • concentration.The radio-enhancing effect of the SPIONs originates from the catalytic activity of their surfaces.X-rays, even at 1 Gy, verifiably destroy the coating of intracellular SPIONs terminated with citrate or malate moieties.X-radiation-induced ablation of the surface layer of the SPIONs creates highly reactive surfaces containing Fe 3+ and Fe 2+ ions that catalyze the ROS formation via the Fenton mechanism and Haber-Weiss cycle.The ROS concentration was found to be increased by more than 300% [16].
Another encouraging approach for radio-enhancing nanomaterials is based on the fusion of plasmonic gold and superparamagnetic Fe 3 O 4 nanoparticles yielding Fe 3 O 4 -Au nanocomposites.In the recent past, sophisticated synthesis strategies for the preparation of Fe 3 O 4 -Au nanocomposites have facilitated the realization of diverse magnetoplasmonic nanoparticles, including core-shell, flower-like and dumbbell-like nanoheterostructures [17].For instance, Fe 3 O 4 @Au core-shell nanoparticles exhibit an encouraging potential for biomedical applications [18][19][20][21][22], since the gold surface provides biocompatibility and, furthermore, may be easily functionalized in aqueous solutions with thiol-containing biomolecules.Unfortunately, the complete gold shell also limits the radio-enhancing performance, because it completely blocks the catalytic activity of the Fe 3 O 4 nanoparticle surface.In contrast, dumbbell-like Au-Fe 3 O 4 nanoheterodimers should be perfectly tailored for their application as radio-enhancing agents, since such nanoheterodimers unify the high-Z material and the Fe 3 O 4 catalyst surface [23,24].However, the major drawback of the Au-Fe 3 O 4 nanodumbbells concerns their surfaces, which are irreversibly stabilized with oleic acid, completely hampering their solubility in aqueous solution.The surfactants oleic acid and oleylamine are essential for the synthesis of Au-Fe 3 O 4 nanodumbbells, which only succeeds through thermal decomposition of an iron precursor (e.g., Fe(CO) 5 , Fe(acac) 3 ) on AuNPs in the presence oleic acid and oleylamine at temperatures above 300 • C [16,[25][26][27].
In this contribution, we report a novel, simple, one-pot synthesis route enabling the preparation of water-soluble, non-toxic patchy Fe 3 O 4 -Au nanoparticles, which exhibit an encouraging radiosensitizer potential for cancer therapy.The patchy surface architecture emerges from growing gold nanoclusters on the Fe 3 O 4 nanoparticle surface.Surface-grown Au nanopatches are expected to enhance the radio-catalytic activity of the Fe 3 O 4 nanoparticles due to interfacial communication [26].X-ray irradiation of intracellular Fe 3 O 4 -Au nanoparticles may result in Fe 3 O 4 surface-catalyzed production of ROS and the generation of O 2 •-near the Au surface, which is significantly enhanced by interfacial electron transfer and excess electronic charges (hot spots) at the Au surface [27].To attain biocompatibility and water solubility, the as-synthesized patchy Fe 3 O 4 -Au nanoparticles (Fe 3 O 4 @Au pNPs) were successfully stabilized with 1-methyl-3-(dodecylphosphonic acid) imidazolium, forming a self-assembled monolayer on the nanoparticle surface.Different kinds of tumor cells (MCF-7 and A549) loaded with Fe 3 O 4 @Au pNPs and exposed to X-radiation in a single dose of 1 Gy were shown to increase the relative ROS concentration by more than 130%.The associated dose-modifying factors (DMF) reached values of 0.448 and 0.422, demonstrating the excellent performance of the Fe 3 O 4 @Au pNPs as X-ray dose-enhancing agents.
Synthesis of the Fe 3 O 4 @Au pNPs.3-Mercaptopropionic acid (MPA) stabilized Fe 3 O 4 @Au pNPs were obtained through alkaline co-precipitation of Fe 3 O 4 NPs in aqueous HAuCl 4 solution, and were subsequently stabilized with 3-mercaptopropionic acid (MPA).21 mL HCl solution was prepared by adding 1 mL of 1 M HCl to 20 mL of water.This solution was heated up to 80 • C under stirring.2 mL of aqueous HAuCl 4 solution (25 mM) were added at once. 3 mL of aqueous ammonium iron(II) sulfate hexahydrate solution (100 mM) and 0.05 mL of aqueous MPA (10 mM) solution were added.After a reaction time of 1 min 3 mL ammonia (30 wt.%) were added.The resulting black suspension was refluxed for 30 min.The resulting MPA-stabilized Fe 3 O 4 @Au pNPs were collected by magnetic decantation and washed thrice with 20 mL ultrapure water.
Surface-coating procedure.For attaining biocompatibility, the initially MPA-stabilized Fe 3 O 4 @Au pNPs and pristine Fe 3 O 4 NPs were coated with 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide (Imidazolium-PA).15 mg of the Fe 3 O 4 @Au pNPs or Fe 3 O 4 NPs were dispersed in water by sonication.Afterwards 40 mM of a methanolic solution of Imidazolium-PA were subjoined, and the mixture was sonicated for 30 min.The Fe 3 O 4 @Au pNPs and Fe 3 O 4 NPs were magnetically isolated and washed thrice.For the Fe 3 O 4 NPs the whole procedure was carried out under argon.
Synthesis of gold nanocluster.MPA-stabilized gold nanoclusters (AuNCs) were prepared by dissolving HAuCl 4 (35.7 µmol) in methanol (7 mL), followed by the addition of MPA (142.8 µmol).The mixture was cooled in an ice bath for 30 min.NaBH 4 dissolved in water (0.2 M, 357 µmol, 0 • C), was then rapidly injected into the AuCl 4 --MPA-mixture under vigorous stirring.The reaction mixture changed its color to dark red-brown under bubbling with nitrogen.After 1 hour of reaction in the ice bath, the mixture was centrifuged (8500 rpm, 10 min).The collected precipitate was subsequently washed with methanol and after the removal of the solvent the precipitate was dried in vacuum and the product was obtained as a black powder.Characterization of the nanoparticles.UV-Vis absorption and Fourier-Transform Infrared (FTIR) spectra were measured using a Perkin Elmer Lambda 2 (Perkin Elmer, Rodgau, Germany, 1987) and a Shimadzu IR Prestige-21 (Shimadzu Europa GmbH, Duisburg, Germany, 2002)) device, respectively.The FTIR spectra were obtained in the attenuated total reflectance (ATR) mode on a diamond/ZnSe crystal plate (MIRacle ATR, Pike Technologies, Madison, WI, USA, 2015).Experiments with energy dispersive X-ray spectroscopy (EDX) were performed using a QUANTAX EDS system (Bruker Nano GmbH, Berlin, Germany, 2012).Transmission electron microscopy (TEM) images were taken with a Zeiss EM 900 TE microscope (Carl Zeiss GmbH, Oberkochen, Germany, 2010).This TE microscope has a nominal point resolution of 5.0 Å at Scherzer defocus and was operated at an acceleration voltage of 80 kV.High resolution transmission electron microscopy (HRTEM) was applied using a Phillips CM 300 UltraTWIN TE microscope (Philips, Eindhoven, The Netherlands, 2000) at an acceleration voltage of 300 kV with a nominal point resolution of 1.7 Å at Scherzer defocus.The EDX spectra were acquired using a Super-X detector.The samples for the TEM und HRTEM investigations were obtained by drop-casting the Fe 3 O 4 @Au pNPs solution on amorphous holey carbon film coated copper TEM grids.The XRD scans were measured in the Bragg-Brentano geometry by means of a Bruker AXS Advance D8 (Bruker Nano GmbH, Berlin, Germany, 2015) X-ray diffractometer using Cu K α radiation (λ Kα = 1.54 Å) at an acceleration voltage of 30 kV.The magnetization curves and ZFC/FC vs. temperature plots of the Fe 3 O 4 @Au pNPs and Fe 3 O 4 NPs were recorded on a Quantum Design MPMS-XL5 SQUID magnetometer (Quantum Design Inc., San Diego, CA, USA, 2015).
Cell experiments.The biocompatibility and the diverse ROS assays were executed by means of a Synergy HT microplate reader (BioTek Inc., Winooski, VT, USA, 2016).TEM images of MCF-7 cells incorporating Fe 3 O 4 @Au pNPs were obtained with a Zeiss EM 906 (Carl Zeiss GmbH, Oberkochen, Germany, 1993).The diverse kinds of cell lines (i.e., MCF-7, MCF-10A and A459) were exposed to X-radiation generated by a 120 kV X-ray tube equipped with a tungsten anode (Comet MXR 160/0.4-3.0,Comet, Flamatt, Switzerland, 2011).The X-radiation has an on average energy value of 34 keV and a maximum energy value of 120 keV.
Neutral red assay.The MCF-7, MCF-10A and A459 cells were seeded in 96-well plates at a cell per well density of 2-3 × 10 4 and were incubated overnight.The cell culture medium was replaced by a medium containing the Fe 3 O 4 @Au pNPs, Fe 3 O 4 NPs or gold nanocluster (AuNC) at a concentration of 10 µg/mL.Subsequently to 24 h incubation, the cell medium was substituted by an FCS-free medium containing 40 µg/mL neutral red (NR).After 2 h incubation at 37 • C, the NR-enriched medium was aspirated off, and the cells were rinsed with PBS.NR molecules trapped inside the lysosomes were released by the addition of 100 µL/well dye-destaining solution (50% ethanol, 49% water, 1% glacial acetic acid).The absorbance of the NR solution was measured at 540 nm.
Cell preparation for TEM examinations.MCF-7 cells were incubated overnight with culture medium containing the Fe 3 O 4 @Au pNPs at a concentration of 10 µg/mL.The cells were washed with ice-cold PBS (pH 7.4), were subsequently fixed with 2.5% glutaraldehyde at 4 • C (overnight) and then post-fixed in 1% OsO 4 and 3% K 3 Fe(CN) 6 at room temperature.Thereafter, the cells were dehydrated in ethanol and encapsulated in Epon.60-70-nm-thick slices were obtained with an ultramicrotome and mounted on Epon blocks.Non-contrasted silver-grey ultrathin slices were imaged.
ROS, superoxide and hydroxyl radical assays.The MCF-7, MCF-10A and A459 cells were seeded in 96-well plates at a cell per well density of 2-3 × 10 4 and were incubated overnight.The cell medium was substituted by one containing the NPs at a concentration of 10 µg/mL.After 24 h incubation, the NP-enriched medium was aspirated off.According to the different assay protocols the respective dye solution was filled in.Subsequently, half of the 96-well plates were treated with X-rays at a single dose of 1 Gy.
ROS assay.2',7'-dichlorofluorescein diacetate (DCFH-DA) was dissolved in DMSO to obtain a 0.01 M stock solution which was diluted with DMEM to 100 µM.The cells were incubated in this solution for 30 min.After cellular uptake, the acetate groups of DCFH-DA were cleaved off so that the DCFH molecules remained inside the cells.Afterwards, the wells were washed with PBS and 100 µL PBS were added in each well.The cells were exposed to X-radiation or remained non-irradiated.ROS oxidize intracellular DCFH to fluorescent DCF.Upon excitation at 480 nm the fluorescence emission of the DCF dye was detected at 528 nm.
Superoxide assay.The relative intracellular concentration of superoxide radicals (O 2 •-) was determined by means of the MitoSOX TM Red mitochondrial superoxide indicator, following the manufacturer's protocol.The dye was dissolved in DMSO at a concentration of 2.5 mM and diluted to 5 µM in Hank's balanced salt solution (HBSS).After 10 min incubation followed by X-ray irradiation, the cells were washed and 100 µL of HBSS were added.The fluorescence of MitoSOX TM Red was excited at 530 nm, and the fluorescence intensity was detected at 590 nm.Hydroxyl radical assay.3'-(p-hydroxyphenyl)-fluorescein (HPF) selectively reacts with HO • .The 5 mM HPF solution was diluted to 5 µM in Krebs-Ringer buffer.After removal of the cell culture medium, 100 µL of HPF solution is added, and the cells were incubated therein for 30 min.Then the cells were washed and exposed to X-radiation.The fluorescence emission of intracellular HPF was excited at 490 nm and detected at 515 nm.
Clonogenic cell survival assay.The MCF-7, MCF-10A and A459 cells were grown in 6-well plates and were incubated in culture medium enriched with the respective kind of NPs (10 µg/mL) overnight.After X-ray irradiation at single dosages of 1, 2 or 3 Gy, the cells were detached, seeded and grown in 6-well plates for 2 weeks to form colonies.The cell colonies were fixed and stained with a mixture of 0.5% (w/v) crystal violet in 50/50 methanol/water for 30 min.Colonies containing more than 50 cells were counted.The colony number was used to calculate the surviving fraction (SF).The survival curves were fitted to a linear quadratic function (ln SF = −(αD + βD 2 )).The X-ray enhancing effect was assessed by determining the dose modifying factor (DMF) from the X-radiation survival curves upon calculating the ratio of radiation doses at the 50% survival level (NP-treated radiation dose divided by the control radiation dose).
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
Patchy Fe 3 O 4 -Au nanoparticles (Fe 3 O 4 @Au pNPs) were obtained through alkaline co-precipitation of Fe 3 O 4 nanoparticles from an aqueous ferrous and ferric solution in presence of Au nanostructures which were formed before through reduction of Au 3+ cations by Fe 2+ cations in acidic solution.The Fe 3 O 4 @Au pNPs were stabilized by subsequent treatment with 3-mercaptopropionic acid (MPA) and 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide (Imidazolium-PA).The formation of the Fe 3 O 4 @Au pNPs was confirmed by examining their composition, structure, morphology and magnetic properties using EDX, XRD, HRTEM, and SQUID.The EDX spectrum of the Fe 3 O 4 @Au pNPs indicates the content of Fe, O and Au in the nanoparticles (Figure 1a).The atomic ratio of Fe to Au is 9:1, which suggests that the Fe 3 O 4 NPs were the main component and formed the core.The powder XRD pattern in Figure 1b  High-resolution transmission electron microscopy (HRTEM) studies, completed by fast Fourier transform (FFT) analyses of the Fe3O4@Au pNPs, provided in-depth insight into the spatial distribution of the chemical composition and crystal phases.The HRTEM images of a Fe3O4@Au pNP in different magnifications (Figure 3) are depicted as a projection along the (511) direction of the Fe3O4 nanocrystal (Figure 3a).The Fe3O4 nanocrystal is partially covered with an Au nanostructure, as it is reflected by the "darker" contrast due to the higher electron density and larger electron-diffracting power of Au in comparison to that of the iron oxide core.The Au composition and structure of the crystalline surface layer were confirmed by the interatomic distance dAu = 0.29 nm, which identifies the crystallographic (111) facet of face-centered cubic gold as having a lattice constant of 0.407 nm (Figure 3b).The distance between two adjacent planes in the Fe3O4 structure is 0.16 nm which is related to the (511) facets of inverse spinel structured Fe3O4 (Figure 3c).These results are consistent with the FFT image analysis of the image phase-contrast variation across the High-resolution transmission electron microscopy (HRTEM) studies, completed by fast Fourier transform (FFT) analyses of the Fe3O4@Au pNPs, provided in-depth insight into the spatial distribution of the chemical composition and crystal phases.The HRTEM images of a Fe3O4@Au pNP in different magnifications (Figure 3) are depicted as a projection along the (511) direction of the Fe3O4 nanocrystal (Figure 3a).The Fe3O4 nanocrystal is partially covered with an Au nanostructure, as it is reflected by the "darker" contrast due to the higher electron density and larger electron-diffracting power of Au in comparison to that of the iron oxide core.The Au composition and structure of the crystalline surface layer were confirmed by the interatomic distance dAu = 0.29 nm, which identifies the crystallographic (111) facet of face-centered cubic gold as having a lattice constant of 0.407 nm (Figure 3b).The distance between two adjacent planes in the Fe3O4 structure is 0.16 nm which is related to the (511) facets of inverse spinel structured Fe3O4 (Figure 3c).These results are consistent with the FFT image analysis of the image phase-contrast variation across the High-resolution transmission electron microscopy (HRTEM) studies, completed by fast Fourier transform (FFT) analyses of the Fe 3 O 4 @Au pNPs, provided in-depth insight into the spatial distribution of the chemical composition and crystal phases.The HRTEM images of a Fe 3 O 4 @Au pNP in different magnifications (Figure 3) are depicted as a projection along the (511) direction of the Fe 3 O 4 nanocrystal (Figure 3a).The Fe 3 O 4 nanocrystal is partially covered with an Au nanostructure, as it is reflected by the "darker" contrast due to the higher electron density and larger electron-diffracting power of Au in comparison to that of the iron oxide core.The Au composition and structure of the crystalline surface layer were confirmed by the interatomic distance d Au = 0.29 nm, which identifies the crystallographic (111) facet of face-centered cubic gold as having a lattice constant of 0.407 nm (Figure 3b).The distance between two adjacent planes in the Fe 3 O 4 structure is 0.16 nm which is related to the (511) facets of inverse spinel structured Fe 3 O 4 (Figure 3c).These results are consistent with the FFT image analysis of the image phase-contrast variation across the Fe 3 O 4 -Au pNP, which reveals 6 diffraction spots for the (111) planes of the Au nanopatch and 2 spots for the (511) planes of the Fe 3 O 4 NP (Figure 3d).The magnetic properties of the Fe3O4@Au pNPs were examined by measuring magnetization curves as a function of the applied magnetic field H and in dependence on the temperature T. The magnetization curve in Figure 4a measured at 300 K reflects the superparamagnetic character of the Fe3O4@Au pNPs.The magnetization saturation (MS) value of the Fe3O4@Au pNPs is 43.6 emu/g.This value is significantly smaller than that (i.e., 66.1 emu/g) derived from the magnetization curves of nearly equally sized Fe3O4 NPs [30].The smaller MS value can be explained by the magnetically inactive gold surface layer.In addition, the interface communication between the Au nanopatches and the Fe3O4 core may also partially destroy the magnetization properties of the Fe3O4 NPs.The superparamagnetic behavior of the Fe3O4@Au pNPs is confirmed by the temperature dependence of the zero-field cooled-field cooled (ZFC-FC) magnetization under an applied magnetic field of 0.01 T (Figure 4b).The ZFC curve exhibits a broad maximum indicating the superparamagnetic blocking temperature at 80 K.The broadness of the ZFC curve indicates the existence of a dipolar coupling between nanoparticles due to their proximal distance.The magnetic properties of the Fe 3 O 4 @Au pNPs were examined by measuring magnetization curves as a function of the applied magnetic field H and in dependence on the temperature T. The magnetization curve in Figure 4a measured at 300 K reflects the superparamagnetic character of the Fe 3 O 4 @Au pNPs.The magnetization saturation (M S ) value of the Fe 3 O 4 @Au pNPs is 43.6 emu/g.This value is significantly smaller than that (i.e., 66.1 emu/g) derived from the magnetization curves of nearly equally sized Fe 3 O 4 NPs [30].The smaller M S value can be explained by the magnetically inactive gold surface layer.In addition, the interface communication between the Au nanopatches and the Fe 3 O 4 core may also partially destroy the magnetization properties of the Fe 3 O 4 NPs.The superparamagnetic behavior of the Fe 3 O 4 @Au pNPs is confirmed by the temperature dependence of the zero-field cooled-field cooled (ZFC-FC) magnetization under an applied magnetic field of 0.01 T (Figure 4b).The ZFC curve exhibits a broad maximum indicating the superparamagnetic blocking temperature at 80 K.The broadness of the ZFC curve indicates the existence of a dipolar coupling between nanoparticles due to their proximal distance.The UV-Vis absorption spectrum of an aqueous solution of MPA-stabilized Fe3O4@Au pNPs (Figure 5) exhibits a broad shoulder between 320 and 420 nm, and a smaller one between 480 and 500 nm.The spectral feature around 490 nm is assigned to the partially masked plasmon resonance band of larger gold nanopatches, whereas the broad shoulder presumably arises from charge transfer across the Au-Fe3O4 interface.To improve the water solubility, the MPA-terminated Fe3O4@Au pNPs were subsequently coated with 1-methyl-3-(dodecylphosphonic acid) imidazolium (Imidazolium-PA).This amphiphilic surfactant was recently shown to form a self-assembled monolayer on a nanoparticle surface [31].The FTIR transmission spectra of the MPA-terminated Fe3O4@Au pNPs (black line) and the Imidazolium-PA-terminated Fe3O4@Au pNPs (red line) are depicted in Figure 6.For MPA-terminated Fe3O4@Au pNPs, the two peaks at 1645 and 1532 cm −1 , which are ascribed to the symmetric and asymmetric carboxylate (COO − ) stretch, respectively, substantiate that MPA was covalently bound to the Fe3O4 nanoparticle surface [32].The absence of the characteristic peak at 2575 cm −1 for the S-H stretching represents an indirect evidence for the formation of the covalent Au-S bond at the gold surface.MPA was very likely bound to both, the Fe3O4 and Au surface.In case of the surfactant Imidazolium-Pa, the predominant peaks at 991 and 1395 cm −1 are assigned to the symmetric P-O and P=O stretching vibration, respectively.Furthermore, the CH2/CH3-stretching The UV-Vis absorption spectrum of an aqueous solution of MPA-stabilized Fe 3 O 4 @Au pNPs (Figure 5) exhibits a broad shoulder between 320 and 420 nm, and a smaller one between 480 and 500 nm.The spectral feature around 490 nm is assigned to the partially masked plasmon resonance band of larger gold nanopatches, whereas the broad shoulder presumably arises from charge transfer across the Au-Fe 3 O 4 interface.The UV-Vis absorption spectrum of an aqueous solution of MPA-stabilized Fe3O4@Au pNPs (Figure 5) exhibits a broad shoulder between 320 and 420 nm, and a smaller one between 480 and 500 nm.The spectral feature around 490 nm is assigned to the partially masked plasmon resonance band of larger gold nanopatches, whereas the broad shoulder presumably arises from charge transfer across the Au-Fe3O4 interface.To improve the water solubility, the MPA-terminated Fe3O4@Au pNPs were subsequently coated with 1-methyl-3-(dodecylphosphonic acid) imidazolium (Imidazolium-PA).This amphiphilic surfactant was recently shown to form a self-assembled monolayer on a nanoparticle surface [31].The FTIR transmission spectra of the MPA-terminated Fe3O4@Au pNPs (black line) and the Imidazolium-PA-terminated Fe3O4@Au pNPs (red line) are depicted in Figure 6.For MPA-terminated Fe3O4@Au pNPs, the two peaks at 1645 and 1532 cm −1 , which are ascribed to the symmetric and asymmetric carboxylate (COO − ) stretch, respectively, substantiate that MPA was covalently bound to the Fe3O4 nanoparticle surface [32].The absence of the characteristic peak at 2575 cm −1 for the S-H stretching represents an indirect evidence for the formation of the covalent Au-S bond at the gold surface.MPA was very likely bound to both, the Fe3O4 and Au surface.In case of the surfactant Imidazolium-Pa, the predominant peaks at 991 and 1395 cm −1 are assigned to the symmetric P-O and P=O stretching vibration, respectively.Furthermore, the CH2/CH3-stretching To improve the water solubility, the MPA-terminated Fe 3 O 4 @Au pNPs were subsequently coated with 1-methyl-3-(dodecylphosphonic acid) imidazolium (Imidazolium-PA).This amphiphilic surfactant was recently shown to form a self-assembled monolayer on a nanoparticle surface [31].The FTIR transmission spectra of the MPA-terminated Fe 3 O 4 @Au pNPs (black line) and the Imidazolium-PA-terminated Fe 3 O 4 @Au pNPs (red line) are depicted in Figure 6.For MPA-terminated Fe 3 O 4 @Au pNPs, the two peaks at 1645 and 1532 cm −1 , which are ascribed to the symmetric and asymmetric carboxylate (COO − ) stretch, respectively, substantiate that MPA was covalently bound to the Fe 3 O 4 nanoparticle surface [32].The absence of the characteristic peak at 2575 cm −1 for the S-H stretching represents an indirect evidence for the formation of the covalent Au-S bond at the gold surface.MPA was very likely bound to both, the Fe 3 O 4 and Au surface.In case of the surfactant Imidazolium-Pa, the predominant peaks at 991 and 1395 cm −1 are assigned to the symmetric P-O and P=O stretching vibration, respectively.Furthermore, the CH 2 /CH 3 -stretching vibrational bands peaking at 2849 and 2920 cm −1 indicate the surface binding of Imidazolium-PA for Fe 3 O 4 @Au pNPs.The cellular uptake of Imidazolium-PA-terminated Fe3O4@Au pNPs by human breast adenocarcinoma cells (MCF-7 cells) was examined using TEM (Figure 7).Apparently, the TEM image of the MCF-7 cell reveals that Fe3O4@Au pNPs formed large agglomerates (black spots) within the cytoplasm.These agglomerates were entrapped in cellular vesicles (endosomes) which indicates cellular uptake via endocytosis.The biocompatibility of the Imidazolium-PA-terminated Fe3O4@Au pNPs was compared with those of Imidazolium-PA-terminated Fe3O4 NPs and MPA-stabilized gold nanoclusters (AuNCs) upon examining their influence on the viability of tumor cells (MCF-7 and adenocarcinomic human alveolar basal epithelial (A459) cells) and healthy cells (human breast epithelial cell line (MCF-10A)) (Figure 8).The cellular uptake of Imidazolium-PA-terminated Fe 3 O 4 @Au pNPs by human breast adenocarcinoma cells (MCF-7 cells) was examined using TEM (Figure 7).Apparently, the TEM image of the MCF-7 cell reveals that Fe 3 O 4 @Au pNPs formed large agglomerates (black spots) within the cytoplasm.These agglomerates were entrapped in cellular vesicles (endosomes) which indicates cellular uptake via endocytosis.The biocompatibility of the Imidazolium-PA-terminated Fe 3 O 4 @Au pNPs was compared with those of Imidazolium-PA-terminated Fe 3 O 4 NPs and MPA-stabilized gold nanoclusters (AuNCs) upon examining their influence on the viability of tumor cells (MCF-7 and adenocarcinomic human alveolar basal epithelial (A459) cells) and healthy cells (human breast epithelial cell line (MCF-10A)) (Figure 8).The cellular uptake of Imidazolium-PA-terminated Fe3O4@Au pNPs by human breast adenocarcinoma cells (MCF-7 cells) was examined using TEM (Figure 7).Apparently, the TEM image of the MCF-7 cell reveals that Fe3O4@Au pNPs formed large agglomerates (black spots) within the cytoplasm.These agglomerates were entrapped in cellular vesicles (endosomes) which indicates cellular uptake via endocytosis.The biocompatibility of the Imidazolium-PA-terminated Fe3O4@Au pNPs was compared with those of Imidazolium-PA-terminated Fe3O4 NPs and MPA-stabilized gold nanoclusters (AuNCs) upon examining their influence on the viability of tumor cells (MCF-7 and adenocarcinomic human alveolar basal epithelial (A459) cells) and healthy cells (human breast epithelial cell line (MCF-10A)) (Figure 8).The respective cell viability data in Figure 8 were obtained by conducting the neutral red assay.The dashed line at 100% represents the viability of the respective control cells.Fe3O4@Au pNPs at a concentration of 10 μg/mL were found to decrease the viability of MCF-7 and MCF-10A cells down to 80%, whereas the A549 cells were hardly impaired.Intracellular AuNCs were found to diminish the viability of all cell lines under study by 10 to 13%.In contrast, the biocompatibility of the Imidazolium-PA-stabilized Fe3O4 NPs is remarkably high, exhibiting cell viability values of around 95% for the MCF-7, MCF-10A and A549 cells.This is explained by the shielding effect of Imidazolium-PA, which is self-assembled in a monolayer on the Fe3O4 NP surface, obviously preventing the formation of HO • at the Fe 2+ cation-containing surface.The slightly toxic effect of the MPA-ligands apparently provided a lower cell viability value for the AuNC and Fe3O4@Au pNPs.
To examine the impact of the Fe3O4@Au pNPs on X-ray-induced formation of the intracellular formation of reactive oxygen species (ROS), MCF-7, A549 and MCF-10A cells were incubated with Fe3O4 NPs, AuNC or Fe3O4@Au pNPs and subsequently exposed to X-radiation at a single dose of 1 Gy (Figures 9 and 10).The change of the intracellular formation of ROS, in total, as well as that of superoxide anion radical (O2 •-) or hydroxyl radical (HO • ), in particular, were quantified using the dichlorofluorescein (DCF) assay, mitochondrial superoxide indicator MitoSox TM Red, and the 3'-(p-hydroxyphenyl)-fluorescein (HPF) assay, respectively.Whereas MCF-7 cells loaded with Fe3O4 NPs or AuNC did not exhibit any change of ROS and HO • formation, intracellular Fe3O4@Au pNPs apparently raised the ROS and HO • levels by 40 and 10%, respectively (Figures 9a and 10a).All kinds of nanoparticles were observed to boost the relative O2 •-concentration by 30 to 40% (Figure 9b).These results are consistent with the cell viability study (Figure 8) and confirm the highest biocompatibility for the Imidazolium-PA-terminated Fe3O4 NPs.X-ray exposure of the MCF-7 cells at a single dose of 1 Gy resulted into a significant enhancement of the ROS formation for all kinds of nanoparticles.While intracellular Fe3O4@Au pNPs provided an increase of the ROS concentration by 120%, the Fe3O4-NPs and AuNC caused an increase of 85% and 60%, respectively (Figure 9a).On the other hand, X-ray irradiation of MCF-7 cells loaded with Fe3O4 NPs provoked O2 •-degradation by 20%, which probably occurs through the X-ray-activated Fe3O4 surfaces as requiring H2O2 and O2 •- consumption during the Fenton and Haber-Weiss reaction (Figure 10b).In comparison, the intracellular AuNC and Fe3O4@Au pNPs mediated an increase of the O2 •-level by 70% and 20%, respectively.This is due to X-ray-induced emission of photoelectrons and Auger electrons from the AuNC and Au nanopatches, which generated O2 •-due to the reduction of nearby oxygen.The HO • formation in MCF-7 cells under X-ray exposure was enhanced by intracellular AuNC by 25%, only, whereas X-ray interactions with intracellular Fe3O4 NP and Fe3O4@Au pNPs raised the HO • concentration for ca.80% (Figure 9c).The latter is explained by X-ray-induced activation of the Fe3O4 surfaces, which catalyze the HO • production through the Fenton mechanism (Figure 11a).The X-ray-provoked impact on the ROS formation in different kinds of cells-MCF-7, A549 and MCF-10A cells-when loaded with Fe3O4@Au pNPs is illustrated in Figure 10b.Apparently, the ROS formation in A549 cells achieved the highest concentration increase, at 140%, whereas Fe3O4@Au The respective cell viability data in Figure 8 were obtained by conducting the neutral red assay.The dashed line at 100% represents the viability of the respective control cells.Fe 3 O 4 @Au pNPs at a concentration of 10 µg/mL were found to decrease the viability of MCF-7 and MCF-10A cells down to 80%, whereas the A549 cells were hardly impaired.Intracellular AuNCs were found to diminish the viability of all cell lines under study by 10 to 13%.In contrast, the biocompatibility of the Imidazolium-PA-stabilized Fe 3 O 4 NPs is remarkably high, exhibiting cell viability values of around 95% for the MCF-7, MCF-10A and A549 cells.This is explained by the shielding effect of Imidazolium-PA, which is self-assembled in a monolayer on the Fe 3 O 4 NP surface, obviously preventing the formation of HO • at the Fe 2+ cation-containing surface.The slightly toxic effect of the MPA-ligands apparently provided a lower cell viability value for the AuNC and Fe 3 O 4 @Au pNPs.
To examine the impact of the Fe 3 O 4 @Au pNPs on X-ray-induced formation of the intracellular formation of reactive oxygen species (ROS), MCF-7, A549 and MCF-10A cells were incubated with Fe 3 O 4 NPs, AuNC or Fe 3 O 4 @Au pNPs and subsequently exposed to X-radiation at a single dose of 1 Gy (Figures 9 and 10).The change of the intracellular formation of ROS, in total, as well as that of superoxide anion radical (O 2 •-) or hydroxyl radical (HO • ), in particular, were quantified using the dichlorofluorescein (DCF) assay, mitochondrial superoxide indicator MitoSox TM Red, and the 3'-(p-hydroxyphenyl)-fluorescein (HPF) assay, respectively.Whereas MCF-7 cells loaded with Fe 3 O 4 NPs or AuNC did not exhibit any change of ROS and HO • formation, intracellular Fe 3 O 4 @Au pNPs apparently raised the ROS and HO • levels by 40 and 10%, respectively (Figures 9a and 10a).All kinds of nanoparticles were observed to boost the relative O 2 •-concentration by 30 to 40% (Figure 9b).These results are consistent with the cell viability study (Figure 8) and confirm the highest biocompatibility for the Imidazolium-PA-terminated Fe 3 O 4 NPs.X-ray exposure of the MCF-7 cells at a single dose of 1 Gy resulted into a significant enhancement of the ROS formation for all kinds of nanoparticles.While intracellular Fe 3 O 4 @Au pNPs provided an increase of the ROS concentration by 120%, the Fe 3 O 4 -NPs and AuNC caused an increase of 85% and 60%, respectively (Figure 9a).On the other hand, X-ray irradiation of MCF-7 cells loaded with Fe 3 O 4 NPs provoked O 2 •-degradation by 20%, which probably occurs through the X-ray-activated Fe 3 O 4 surfaces as requiring H 2 O 2 and O 2 •consumption during the Fenton and Haber-Weiss reaction (Figure 10b).In comparison, the intracellular AuNC and Fe 3 O 4 @Au pNPs mediated an increase of the O 2 •-level by 70% and 20%, respectively.This is due to X-ray-induced emission of photoelectrons and Auger electrons from the AuNC and Au nanopatches, which generated O 2 •-due to the reduction of nearby oxygen.The HO • formation in MCF-7 cells under X-ray exposure was enhanced by intracellular AuNC by 25%, only, whereas X-ray interactions with intracellular Fe 3 O 4 NP and Fe 3 O 4 @Au pNPs raised the HO • concentration for ca.80% (Figure 9c).The latter is explained by X-ray-induced activation of the Fe 3 O 4 surfaces, which catalyze the HO • production through the Fenton mechanism (Figure 11a).The X-ray-provoked impact on the ROS formation in different kinds of cells-MCF-7, A549 and MCF-10A cells-when loaded with Fe 3 O 4 @Au pNPs is illustrated in Figure 10b.Apparently, the ROS formation in A549 cells achieved the highest concentration increase, at 140%, whereas Fe 3 O 4 @Au pNPs in MCF-10A cells enhanced the ROS formation by only 30%.These results are consistent with the observed increases of the HO • level in A549 and MCF-10A cells by 100% and 30%, respectively.On the other hand, the O 2 •-production in A549 and MCF-10A cells containing Fe 3 O 4 @Au pNPs appeared to be hardly altered.
Appl.Sci.2018, 8, x FOR PEER REVIEW 11 of 16 pNPs in MCF-10A cells enhanced the ROS formation by only 30%.These results are consistent with the observed increases of the HO • level in A549 and MCF-10A cells by 100% and 30%, respectively.On the other hand, the O2 •-production in A549 and MCF-10A cells containing Fe3O4@Au pNPs appeared to be hardly altered.pNPs in MCF-10A cells enhanced the ROS formation by only 30%.These results are consistent with the observed increases of the HO • level in A549 and MCF-10A cells by 100% and 30%, respectively.On the other hand, the O2 •-production in A549 and MCF-10A cells containing Fe3O4@Au pNPs appeared to be hardly altered.pNPs in MCF-10A cells enhanced the ROS formation by only 30%.These results are consistent with the observed increases of the HO • level in A549 and MCF-10A cells by 100% and 30%, respectively.On the other hand, the O2 •-production in A549 and MCF-10A cells containing Fe3O4@Au pNPs appeared to be hardly altered.The clonogenic cell survival assay was employed to survey the impact of the Fe3O4 NPs, AuNC or Fe3O4@Au pNPs on the survival and proliferation of the MCF-7, A549 and MCF-10A cells exposed to X-radiation at single doses of 1, 2, and 3 Gy (Figure 11b,c,d).As expected, the survival curves of irradiated MCF-7 and A549 cells loaded with the diverse kinds of NPs exhibited significantly faster decays than those of the control cells (medium).Amazingly, the AuNC and Fe3O4 NPs coincided in their influence on the survival behavior of both tumor cell lines, although they enhanced the X-ray-induced generation of two different kinds of ROS.The survival curves obtained for the Fe3O4@Au pNP loaded MCF-7 and A549 cells (blue) showed the steepest decays, which are consistent with the associated dose-modifying factor (DMF) values of 0.448 and 0.422 (Table 1).These DMF values are considerably lower than those obtained for the AuNC and Fe3O4 NP-loaded MCF-7 and A549 cells, which were 0.611 and 0.616, respectively.In contrast, the survival curves of the healthy cells (MCF-10A), when incorporating either Fe3O4@Au pNPs or Fe3O4 NPs, display similar weak decreases and relatively large DMF values of 0.804 and 0.731, whereas intracellular AuNC caused a more quickly decaying survival curve and a rather small DMF value of 0.344.

Discussion
Water-soluble, biocompatible patchy Fe3O4-Au nanoparticles (Fe3O4@Au pNPs) were prepared through a facile one-pot synthesis procedure followed by functionalization with Imidazolium-PA forming a positively charged, self-assembled monolayer on the surface [31].The one-pot synthesis route started with a redox reaction in acidic solution and was followed by co-precipitation under alkaline conditions.The initial redox reaction between Au 3+ and Fe 2+ cations did not only produce elemental Au 0 and thereupon, Au nuclei but also Fe 3+ cations which fed the subsequent alkaline co-precipitation process.In the supersaturated acidic reaction solution, AuNCs were formed.The subsequent alkaline co-precipitation provided the formation of Fe3O4 NPs in the presence of AuNC which epitaxially grew on the surface of the Fe3O4 NPs, probably under prosecution of the redox reaction.The morphology, composition and surface chemistry, crystallographic, magnetic and The clonogenic cell survival assay was employed to survey the impact of the Fe 3 O 4 NPs, AuNC or Fe 3 O 4 @Au pNPs on the survival and proliferation of the MCF-7, A549 and MCF-10A cells exposed to X-radiation at single doses of 1, 2, and 3 Gy (Figure 11b,c,d).As expected, the survival curves of irradiated MCF-7 and A549 cells loaded with the diverse kinds of NPs exhibited significantly faster decays than those of the control cells (medium).Amazingly, the AuNC and Fe 3 O 4 NPs coincided in their influence on the survival behavior of both tumor cell lines, although they enhanced the X-ray-induced generation of two different kinds of ROS.The survival curves obtained for the Fe 3 O 4 @Au pNP loaded MCF-7 and A549 cells (blue) showed the steepest decays, which are consistent with the associated dose-modifying factor (DMF) values of 0.448 and 0.422 (Table 1).These DMF values are considerably lower than those obtained for the AuNC and Fe 3 O 4 NP-loaded MCF-7 and A549 cells, which were 0.611 and 0.616, respectively.In contrast, the survival curves of the healthy cells (MCF-10A), when incorporating either Fe 3 O 4 @Au pNPs or Fe 3 O 4 NPs, display similar weak decreases and relatively large DMF values of 0.804 and 0.731, whereas intracellular AuNC caused a more quickly decaying survival curve and a rather small DMF value of 0.344.

Discussion
Water-soluble, biocompatible patchy Fe 3 O 4 -Au nanoparticles (Fe 3 O 4 @Au pNPs) were prepared through a facile one-pot synthesis procedure followed by functionalization with Imidazolium-PA forming a positively charged, self-assembled monolayer on the surface [31].The one-pot synthesis route started with a redox reaction in acidic solution and was followed by co-precipitation under alkaline conditions.The initial redox reaction between Au 3+ and Fe 2+ cations did not only produce elemental Au 0 and thereupon, Au nuclei but also Fe 3+ cations which fed the subsequent alkaline co-precipitation process.In the supersaturated acidic reaction solution, AuNCs were formed.The subsequent alkaline co-precipitation provided the formation of Fe 3 O 4 NPs in the presence of AuNC which epitaxially grew on the surface of the Fe 3 O 4 NPs, probably under prosecution of the redox reaction.The morphology, composition and surface chemistry, crystallographic, magnetic and optical properties of the Fe 3 O 4 @Au pNPs were characterized using HRTEM, EDX, and FTIR transmission spectroscopy, XRD, SQUID, and UV-Vis absorption spectroscopy, respectively.
As is evident from the XRD data, HRTEM and EDX analysis, the 12.5 nm-sized Fe 3 O 4 @Au pNPs consist of a spherical monocrystalline Fe 3 O 4 core that is partially coated with crystalline AuNCs and Au nanopatches (Figure 3).Consistently, the Fe 3 O 4 @Au pNPs were found to exhibit superparamagnetism, with a Ms value of 43.6 emu/g, which is smaller than that of nearly equally sized Fe 3 O 4 NPs [30].This difference is thought to arise from the interface communication between the Au nanopatches and the Fe 3 O 4 core, which partially degrades the magnetization.This is corroborated by the quenching of the surface plasmon resonance band in the UV-Vis absorption spectrum (Figure 5), as indicating interfacial electron transfer from the Au 0 to Fe 3+ cations.As is obvious from the TEM image (Figure 7) displaying agglomerates in endosomes, internalization by MCF-7 cells followed the endocytic pathway.The impact of the different kinds of nanoparticles on the viability of the cancerous MCF-7 and A549 cells and healthy epithelial MCF-10A cells was evaluated by performing the neutral red assay (Figure 8).Imidazolium-PA-stabilized Fe 3 O 4 @Au pNPs at a concentration of 10 µg/mL degraded the viability of MCF-7 and MCF-10A cells by 20%, whereas the A549 cells were hardly damaged.Intracellular AuNC were observed to decrease the viability of MCF-7, A549 and MCF-10A cells to values between 87% and 90%.In contrast, the biocompatibility of the Imidazolium-PA-stabilized Fe 3 O 4 NPs is remarkably high, exhibiting cell viability values of around 95% for all kinds of cells under study.This is due to the shielding effect of Imidazolium-PA, which is self-assembled in a monolayer on the Fe 3 O 4 NP surface, obviously preventing the formation of HO • at the Fe 2+ cation-containing surface.The slightly higher cytotoxicity effect of the Fe 3 O 4 @Au pNPs is explained with the restrained adsorption of the Imidazolium-PA surfactants at the initially MPA-terminated gold nanopatches.The MPA ligands apparently provided lower cell viability values for both the AuNCs and the Fe 3 O 4 @Au pNPs.The performance of the Fe 3 O 4 @Au pNPs as X-ray dosage enhancers was compared with that of the AuNCs and Fe  [27].The influence of the Fe 3 O 4 @Au pNPs on X-ray-induced ROS formation in the tumor MCF-7 and A549 cells was compared with that in healthy MCF-10A cells.Apparently, the ROS formation in A549 cells containing the Fe 3 O 4 @Au pNPs reached the highest concentration increase being 140%, whereas Fe 3 O 4 @Au pNPs in X-ray irradiated MCF-10A cells enhanced ROS formation by 30% only.These results are consistent with the observed increases of the HO • level in A549 and MCF-10A cells of 100% and 30%, respectively.On the other hand, the X-radiation-induced O 2 •-production in A549 and MCF-10A cells loaded with Fe 3 O 4 @Au pNPs appeared to be hardly altered.All these results indicate that the effect of Fe 3 O 4 @Au pNPs on the X-ray enhancement of the ROS production in tumor cells is considerably larger than that in healthy MCF-10A cells.
Appl.Sci.2018, 8, x FOR PEER REVIEW 7 of 16Fe3O4-Au pNP, which reveals 6 diffraction spots for the (111) planes of the Au nanopatch and 2 spots for the (511) planes of the Fe3O4 NP (Figure3d).

Figure 3 .
Figure 3. (a) HRTEM image of a Fe 3 O 4 @Au pNP (b,c) with magnified sections, and (d) the fast Fourier transform (FFT) image.

Figure 4 .
Figure 4. (a) Magnetization curve at 300 K and, (b) the temperature dependence of the ZFC/FC magnetization of the Fe3O4@Au pNPs.

Figure 4 .
Figure 4. (a) Magnetization curve at 300 K and, (b) the temperature dependence of the ZFC/FC magnetization of the Fe 3 O 4 @Au pNPs.

Figure 4 .
Figure 4. (a) Magnetization curve at 300 K and, (b) the temperature dependence of the ZFC/FC magnetization of the Fe3O4@Au pNPs.

Figure 6 .
Figure 6.FTIR transmission spectra of the Fe3O4@Au pNPs and the structure formula of the surfactants MPA (top right) and Imidazolium-PA (bottom right).

Figure 6 .
Figure 6.FTIR transmission spectra of the Fe 3 O 4 @Au pNPs and the structure formula of the surfactants MPA (top right) and Imidazolium-PA (bottom right).

Figure 6 .
Figure 6.FTIR transmission spectra of the Fe3O4@Au pNPs and the structure formula of the surfactants MPA (top right) and Imidazolium-PA (bottom right).

3 O 4 Fe 3 O 4 @
. Therefore, MCF-7, A549 and MCF-10A cells were incubated with the different kinds of nanoparticles and were exposed to X-radiation with single dosages of 1 Gy, 2 Gy, or 3 Gy.The change of the intracellular formation of ROS, in total, and the alteration of the individual O 2•-and HO • productions were determined upon performing the DCF assay, MitoSox TM Red, and HPF assay, respectively.X-ray irradiation of the MCF-7 cells at 1 Gy significantly enhanced the ROS formation for all kinds of nanoparticles.Intracellular Fe 3 O 4 @Au pNPs increased the ROS concentration by 120%.On the other hand, the Fe 3 O 4 -NPs and AuNCs only caused increases of 85% and 60%, respectively.Intracellular Fe 3 O 4 NPs and AuNC were observed to preferentially enhance either the HO • or the O 2 •-formation in MCF-7 cells only.In comparison, Au pNPs elevated the concentration of both O 2 •-and HO • in MCF-7 and A549 cells, which indicates the synergistic interplay between the Fe 3 O 4 and Au surfaces for X-ray-induced ROS formation.Moreover, efficient electron transfer across the Au-Fe 3 O 4 interface potentiates the catalytic activity of the Fe 3 O 4 surface by reducing surface Fe 3+ to Fe 2+ cations •-concentration in MCF-7 cells loaded with Fe 3 O 4 NPs, AuNC or Fe 3 O 4 @Au pNPs which were exposed to X-radiation at a single dose of 1 Gy or left non-irradiated (n = 6, * p < 0.05, *** p < 0.001, **** p < 0.0001).

Table 1 .
DMF values obtained from the radiation dose survival curves of MCF-7, A549 and MCF-10A cells depicted in Figure10.

Table 1 .
DMF values obtained from the radiation dose survival curves of MCF-7, A549 and MCF-10A cells depicted in Figure10.