Functionalized Au-Fe

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Biological and Medical Applications of Materials and Interfaces 4

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NOBF-Functionalized Au-FeO Nanoheterodimers for Radiation Therapy: Synergy Effect due to Simultaneous Reactive Oxygen and Nitrogen Species Formation Stefanie Klein, Christina Harreiss, Christina Menter, Julian Hümmer, Luitpold V. R. Distel, Karsten Meyer, Rainer Hock, and Carola Kryschi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03660 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

NOBF4-Functionalized Au-Fe3O4 Nanoheterodimers for Radiation Therapy: Synergy Effect due to Simultaneous Reactive Oxygen and Nitrogen Species Formation Stefanie Kleina, Christina Harreißa, Christina Mentera, Julian Hümmerb, Luitpold V. R. Distelc, Karsten Meyerb, Rainer Hockd and Carola Kryschia* aDepartment

of Chemistry and Pharmacy, Physical Chemistry I and ICMM, Friedrich-

Alexander University of Erlangen, Egerlandstr.3, D-91058 Erlangen, Germany. bDepartment

of Chemistry and Pharmacy, Inorganic and General Chemistry,

Friedrich-Alexander University of Erlangen, Egerlandstr. 1, D-91058 Erlangen, Germany. cDepartment

of Radiation Oncology, Friedrich-Alexander University of Erlangen,

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

of Physics, Institute of Crystallography and Structural Physics, Friedrich-

Alexander University of Erlangen, Staudtstr. 3, D-91058 Erlangen, Germany.

ABSTRACT: Snowman-shaped Au-Fe3O4 nanoheterodimers were synthesized by thermal decomposition of iron oleate on pre-synthesized Au nanoparticles. Subsequently performed ligand exchange with nitrosyl tetrafluoroborate provided water solubility and enabled X-ray induced NO release. These Au-Fe3O4 nanoheterodimers combine high-Z material with catalytically

active

Fe3O4

surfaces

and

moreover,

plasmonic

properties

with

superparamagnetic performance. We could establish synergetic interactions between Xradiation and both, the Au and Fe3O4 surfaces, which resulted in the simultaneous production of the nitric oxide radical at the Fe3O4 surface and the superoxide radical at the Au surface. The surface-confined reaction between these radicals generated peroxynitrite. This highly reactive species may cause nitration of mitochondrial proteins, lipid peroxidation, and induce DNA strand breaks. Therefore, high concentrations of peroxynitrite are expected to give rise to severe cellular energetic derangements and thereupon, entail rapid cell death. As providing a common platform for X-ray induced formation of the highly reactive radical nitric oxide, superoxide and peroxynitrite, nitrosyl tetrafluoroborate functionalized Au-Fe3O4 nanosnowmen were shown to exhibit excellent performance as X-ray enhancing agents in radiation therapy.

KEYWORDS:

Au-Fe3O4 nanoheterodimers, nitrosyl tetrafluoroborate coating, radiation

therapy, reactive oxygen species, reactive nitrogen species

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INTRODUCTION In recent past intensive research activities were focused on the design and synthesis of magnetoplasmonic

nanoheterostructures

comprising

superparamagnetic

iron

oxide

nanoparticles (SPIONs) and plasmonic noble metal nanostructures combined in core-shell, onion, dumbbell, flower-like structures.1-3 Whereas the individual nanomaterial components exhibit different optical, electromagnetic, and magnetic properties, their fusion generates novel interfacial electronic and electromagnetic interactions which modifies the redox potential and thus, may significantly improve the catalytic performance of the nanoheterostructure. In particular, nanoheterodimers (NHDs) offer two surfaces that differ in structure and composition and provide therefore specific sites for functional groups which are suited for diagnostic and therapeutic applications.4-10 Dumbbell- and snowman-shaped Au–Fe3O4 NHDs are composed of coalesced gold (Au) and magnetite (Fe3O4) nanospheres with equal or different diameters. Both nanomaterials are biocompatible and exhibit unique advantageous properties, consisting in surface plasmon resonance for gold and superparamagnetism for Fe3O4, which have inspired the design of versatile biomedical applications.3-8,10-11 Typically, Au-Fe3O4 nanodumbbells and nanosnowmen are obtained by epitaxial growing Fe3O4 onto a presynthesized Au nanosphere. The lattice mismatch between both crystalline components causes strain, whereas the different electronic band structures should facilitate electron transfer across the interface. In case of Au-Fe3O4 NHDs, as combining a plasmonic nanosphere with a highly reactive Fe3O4 surface, may significantly enhance the impact of X-rays on tumor tissue. Gold nanoparticles (AuNPs) were shown to perform as X-ray sensitizer due to their large photoelectric absorption coefficient.7,8,12 Intracellular AuNPs, when exposed to keV X-radiation, emit photoelectrons, Auger electrons, and fluorescent X-rays.7 Whereas the emission of secondary electrons results in DNA damage and gives rise to the generation of superoxide anions (O2•-) near the AuNP surface, the fluorescent X-rays produce hydroxyl radicals (HO•).1316

On the other hand, the effect of X-rays on intracellular Fe3O4 nanoparticles (SPIONs)

consists in the creation of highly reactive surfaces due to freely accessible Fe2+ and Fe3+ ions.17-18 In the cytoplasm X-ray activated SPION surfaces were demonstrated to catalyze the Fenton reaction and the Haber-Weiss cycle. This implies that SPIONs boost the impact of Xrays on cells by increasing the formation of reactive oxygen species (ROS) including HO and hydrogen peroxide.17-18 Reactive nitrogen species (RNS) such as nitric oxide (NO•) or peroxynitrite (ONOO-) can evoke nitration of mitochondrial proteins and may also cause oxidative damage to biomolecules comprising tyrosine residues, DNA and phospholipids.19 The ROS and RNS are classified under the category RONS (reactive oxygen and nitrogen species). Primary RONS,


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such as O2-, H2O2 or NO•, are well controlled by superoxide dismutase, catalase and NO synthases so that their reactions with biomolecules are reversible. On the other hand, the secondary RONS (e.g. HO, ONOO-, HOCl) are formed through the interaction between two reactive species and exert deleterious effects on diverse cell functions.19-22 ONOO- is formed by a diffusion-controlled reaction of NO• with O2•-. This highly reactive species may damage a broad variety of biomolecules by oxidation, nitrosylation and nitration reactions. For instance, it causes tyrosine nitration und thereupon, blocks the respective signaling cascades.19,23-26 Among others, ONOO- nitrates mitochondrial proteins, induces lipid peroxidation and DNAstrand breaks. Peroxynitrite at low concentrations leads to programmed cell death (apoptosis). On the other hand, peroxynitrite at sufficiently high concentrations gives rise to severe cellular energetic derangements which entails rapid necrosis.24-26 In this contribution, we report of snowman-shaped Au-Fe3O4 NHDs which were prepared by thermal decomposition of iron (III) oleate on pre-synthesized AuNPs at 320°C in the presence of pre-existing gold nanospheres, oleic acid and oleylamine. The oleicacid/oleylamine terminated Au-Fe3O4 NHDs were transferred into the aqueous phase using nitrosyl tetrafluoroborate (NOBF4) as phase-transfer agent and secondary ligand. X-ray irradiation of intracellular Au-Fe3O4 NHDs, when containing NO+ in their surface structures, was observed to result into the formation of peroxynitrite. Breast cancer cells (MCF-7), loaded with Au-Fe3O4 NHDs and exposed to X-rays at a dosage of 1 Gy, show an increase of the peroxynitrite level for 300 %. As expected from the decomposition of peroxynitrite into HO• and NO2 the rise of the peroxynitrite concentration was observed to be associated with drastically increased membrane lipid peroxidation (MLP) and DNA fragmentation, which are characteristic features for apoptotic cell death.

RESULTS AND DISCUSSION Characterization of the snowman-shaped Au-Fe3O4 NHDs. Snowman-shaped AuFe3O4 NHDs were obtained by thermal decomposition of iron (III) oleate on pre-synthesized AuNPs.27 The AuNPs were synthesized in chloroform by reducing HAuCl4 with tert-butylamine borane in the presence of oleylamine.28 The size of the AuNPs was adjusted to a mean value of 6 nm by tuning the HAuCl4/oleylamine ratio.29 The morphology of the Au-Fe3O4 NHDs was found to depend on both, the concentration of iron (III) oleate and the size of the presynthesized AuNPs. The morphology and size distribution of the Au-Fe3O4 NHDs were examined using transmission electron microscopy (TEM). The TEM image in Figure 1a clearly illustrates the snowman-like shape of the Au-Fe3O4 NHDs. The histogram analysis of the size distribution of the Au-Fe3O4 NHDs yielded a mean size of 11 ± 1.3 nm (see supporting information (SI) Figure S1). The iron and gold contents of the Au-Fe3O4 NHDs was determined ACS Paragon Plus Environment


using inductively coupled plasma-optical emission spectrometry (ICP-OES) and were 76.5 % for iron and 23.5 % for gold. This implies that the iron-to-gold mass ratio in Au-Fe3O4 NHDs is 3.25:1 and thus larger than that of the starting materials with 2.5:1.

(b)

(a)

Figure 1. TEM image (a) and (b) XRD pattern of the snowman-shaped Au-Fe3O4 NHDs.

The crystal structure of the Au-Fe3O4 NHDs was investigated by performing XRD measurements. The powder XRD pattern in Figure 1b displays the characteristic peaks of both, Fe3O4 in the cubic inverse spinel structure and gold in the face centered cubic phase. The peaks at 2 = 30.1°, 35.5°, 43.1°, 53.5°, 57.0° and 62.7° are assigned to diffraction from the (220), (311), (400), (422), (511), and (440) planes of Fe3O4, whereas the diffraction peaks at 38.5°, 44.7°, 65.2°, 78.3° and 82.4° are ascribed to the (111), (200), (220), (311), and (222) planes of gold. The relatively broad widths (FWHM > 0.7°) of the diffraction peaks are consistent with the ultra-small sizes of the Fe3O4 and Au nanostructures. Thus, the XRD pattern confirms the coexistence of crystalline gold and SPIONs as components of the AuFe3O4 NHDs. The UV/Vis absorption spectrum of the Au-Fe3O4 NHDs ((SI): Figure S2) displays a surface plasmon resonance band being red-shifted by 30 nm and broadened by 20 % in comparison with that of 6 nm-sized AuNPs. The red-shift and broadening of the surface plasmon resonance band are explained with efficient electron transfer occurring across the Au-Fe3O4 interface which is rationalized as a spill-out of the free electron-density of the plasmonic AuNP.30 Surface-coating of the Au-Fe3O4 NHDs with NOBF4. Medical and biological applications require the water solubility of Au-Fe3O4 NHDs. For this reason, the oleic acid/oleylamine coating of the NHDs was modified by partial or full exchange against watersoluble ligands. A n-hexane dispersion of the Au-Fe3O4 NHDs was mixed with an acetonitril ACS Paragon Plus Environment

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solution of NOBF4, and partial ligand exchange took place at the two-phase boundary layer ((SI): Figure S3). The NOBF4-stabilized Au-Fe3O4 NHDs were isolated by precipitation with toluene and were finally dispersed in water.31 The surface structures of the NOBF4-stabilized Au-Fe3O4 NHDs were elucidated using FTIR transmission spectroscopy. Figure 2a depicts the FTIR transmission spectra of NOBF4 and NOBF4-stabilized Au-Fe3O4 NHDs. The FTIR spectrum of NOBF4 contains a prominent peak around 990 cm-1 which is assigned to the symmetric B-F stretching vibration31,32, whereas the NO stretching vibration gives rise to a peak around 2200 cm-1.31.33 As expected, NOBF4 bound at the surface of the NHDs exhibits less (b)

(a) 110

30

100

Au-Fe3O4 NHDs in hexane Au-Fe3O4 NHDs in PBS

25

90 80

20

70

counts

transmittance

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60 50 40

Au-Fe3O4 NHDS coated with NOBF4

30

pure NOBF4

15 10 5

20

0

10 500

1000

1500

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2500

3000

10

wavenumber [nm]

20

30

40

50

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size [nm]

Figure 2. (a) FTIR transmission spectra of NOBF4 and NOBF4 stabilized Au-Fe3O4 NHDs, (b) DLS measurements of oleic acid/oleylamine coated Au-Fe3O4 NHDs in n-hexane compared to ththe NOBF4 stabilized Au-Fe3O4 NHDs in PBS buffer. prominent peaks for the B-F vibrational stretching (1050 cm-1)31,32 and the NO vibrational stretching mode (2165 cm-1)31,33. The spectral shift of both peaks, and in particular, the large decrease in peak intensity of the B-F stretching vibration presumably originates from strong electrostatic interactions of the BF4- anions and NO+ cations with the Fe3O4 surface. The additional vibrational peaks in the FTIR spectrum of the Au-Fe3O4 NHDs, in particular the (CH)-stretching vibrations34 at 2925 and 2850 cm-1, are attributed to residual oleylamine and oleic-acid ligands ((SI): Figure S4). These results indicate that oleylamine and oleic acid were either partially replaced by NOBF4 or this ligand was incorporated in the oleic acid/oleylamine shell. The DLS measurements (Figure 2b) were performed to examine how well the oleic acid/oleylamine coated and the NOBF4 stabilized Au-Fe3O4 NHDs may be dispersed in a nonpolar solvent and aqueous solution, respectively. The oleic acid/oleylamine coated AuACS Paragon Plus Environment


Fe3O4 NHDs (black line) in n-hexane possess a narrow size distribution centered at 20 nm. The size distribution of NOBF4 stabilized Au-Fe3O4 NHDs (red line) in phosphate buffered saline (PBS) is slightly broader and has a mean hydrodynamic diameter of 23 nm. Apparently, the NOBF4 stabilized Au-Fe3O4 NHDs exhibit excellent dispersibility in cell media and are suited for biomedical applications. Their large zeta potential value of +40 mV provides strong repulsive electrostatic interactions between the Au-Fe3O4 NHDs and attractive interactions with water. The magnetization curve at 300 K of the NOBF4 stabilized Au-Fe3O4 NHDs ((SI): Figure S5) exhibits no hysteresis and thereupon, reflects superparamagnetism. The value of the saturation magnetization with 30.5 emu/g is significantly smaller than that of SPIONs (74-77 emu/g).18 The considerably smaller saturation magnetization value arises from the nonmagnetic gold component of the Au-Fe3O4 NHDs which contributes at least one half to the total mass of the NHDs. The SPION sizes smaller than 10 nm as well as their bulky surface structures are further reasons for the relatively small saturation magnetization value. RONS Generation after Irradiation. In order to examine the synergetic interplay between the gold and SPION component in Au-Fe3O4 NHDs for their application as X-ray enhancing agent in cancer therapy, the radio-sensitizing effect of NOBF4 stabilized Au-Fe3O4 NHDs were compared to that of NOBF4 stabilized SPIONs in MCF-7 cells under X-ray irradiation. For the sake of comparison the SPIONs were also synthesized following the same synthesis procedure that had been employed for the preparation of the Au-Fe3O4 NHDs. The biocompatibility of the Au-Fe3O4 NHDs and SPIONs was investigated by performing the neutral red cytotoxicity assay on MCF-7 cells. The cell viability was found to be slightly more diminished for intracellular Au-Fe3O4 NHDs than for SPIONs ((SI): Figure S6). The cellular uptake and location of the Au-Fe3O4 NHDs in MCF-7 cells was studied using TEM. The TEM image ((SI): Figure S7) visualizes that the Au-Fe3O4 NHDs had accumulated in the cytoplasm of the MCF-7 cell. For the radiation experiments MCF-7 cells loaded with Au-Fe3O4 NHDs or SPIONs were irradiated with X-rays at a single dose of 1 Gy or left non-irradiated. The X-radiation induced change of the ROS formation was measured using the 2’,7’-dichlorofluorescein (DCF) assay (Figure 3a). All results obtained from the DCF assay and the other radical detection assays are represented as percentage values that are related to either the radical level of nonirradiated or irradiated MCF-7 cells in nanoparticle-free cell culture medium (control cells). The reference values are depicted as a dotted line in the diagrams. Under X-ray exposure intracellular Au-Fe3O4 NHDs were found to raise the ROS formation in the MCF-7 cells above 200 %, while intracellular SPIONs induced a much smaller increase of the ROS level (Figure 3a). Recently Klein et al. reported that SPIONs with the initial Fe3O4 composition gradually lose their performance as X-ray enhancing agent because of the oxidation of surface Fe2+ to Fe3+


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ions.13 In contrast, the Au-Fe3O4 NHDs, despite their long-term storage under ambient conditions, were not observed to undergo any ageing process.

(b)

250

non-irradiated irradiated

200 150 100 50 0

SPION

Au-Fe3O4 NHDs

increase of fluorescence (% of control)


(a)


150

100

50

0

SPION

Au-Fe3O4 NHDs

(c)


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


200


150 100 50 0

superoxide

OH radical

Figure 3. The change in relative concentrations of the ROS (a) and superoxide (b) in MCF-7 cells loaded with SPIONs or Au-Fe3O4 NHDs and (c) the increases of the relative hydroxyl or superoxide radical level in AuNPs loaded MCF-7 cells, when exposed to X-rays with a single dose of 1 Gy or left non-irradiated.

Intracellular AuNPs, when exposed to keV X-radiation, emit photoelectrons, Auger electrons and fluorescent X-rays, but also may achieve X-ray activated surfaces. The emission of secondary electrons causes the formation of hydroxyl radicals (HO•), whereas the X-ray induced catalytic activity of AuNPs results in electron transfer to adsorbed O2 so that superoxide anions (O2•-) will be created. In case of sufficiently small AuNPs (< 6 nm) their large surface curvature facilitates electron spill-out to nearby O2 molecules and thereupon, efficient reduction to O2•-.15 These findings are consistent with results obtained from X-ray irradiated MCF-7 cells containing citrate-stabilized AuNPs (Figure 3c). For comparison, irradiated MCF7 cells loaded with SPIONs or Au-Fe3O4 NHDs, were observed to develop a decrease of the O2- concentrations by 20 % and 40 %, respectively (Figure 3b), whereas the O2- level in nonirradiated cells loaded with these nanoparticles is increased by 20 - 40 %. The increase of the O2- level in non-irradiated MCF-7 cells is understood to arise from adsorption of O2- molecules at positively charged AuNP- and Fe3O4-surface structures which protect them against ACS Paragon Plus Environment


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conversion by superoxide dismutase (SOD) to H2O2. X-ray exposure of intracellular SPIONs or Au-Fe3O4 NHDs generates highly reactive Fe3O4 surfaces which initiates the redox reaction between surface Fe3+ ions and adsorbed O2- to Fe2+ and O2 (Eq.(1)) as well as may support the Fenton reaction (Eq.(2)) that produces HO• radicals. The intracellular HO• radical formation can be detected using the 3´-(p-hydroxyphenyl) fluorescein (HPF) dye assay ((SI): Figure S9). Since the HPF dye also reacts with peroxynitrite, these results demonstrate an increase of both, the HO• radical and peroxynitrite. X-ray exposure of NOBF4-stabilized SPIONs and AuFe3O4 NHDs may also induce Fe3O4-surface mediated electron transfer to the NO+ cations. The resulting NO radicals scavenge O2- by forming peroxynitrite (Eq.(3)) which should also contribute to the observed decrease of the O2- level in X-ray irradiated MCF-7 cells containing NOBF4-stabilized SPIONs and Au-Fe3O4 NHDs. Since O2- is short-living, the formation of peroxynitrite probably takes place near the gold surface of the Au-Fe3O4 NHDs where O2•- will be formed by electron transfer from X-ray activated AuNP surfaces to nearby O2 molecules. Fe3+ + O2- ↔ Fe2+ + O2

(1)

Fe2+ + H2O2 → Fe3+ + OH- + O2-

(2)

NO• + O2- → ONOO-

(3)

Complementary studies employing nitric oxide and peroxynitrite assays were performed, in order to confirm the observed X-ray induced formation of NO radicals in MCF-7 cells with NOBF4 stabilized SPIONs and Au-Fe3O4 NHDs. As a control group the results of these assays are also shown for the citrate-stabilized AuNP. In cells loaded with the AuNPs the level of the NO radical is not altered and the formation of the peroxynitrite is only slightly increased because of the enhancement of the superoxide level, which may increase the intracellular peroxynitrite production (Figure 4). Surface modification with NOBF4 is suggested to result into electrostatic adsorption of the BF4- and NO+ ions at the Au and Fe3O4 surfaces which simultaneously prevents NO+ cations from being hydrolyzed to yield nitrous acid.35 The formation of NO radicals took presumably place at the Au-Fe3O4 NHD and SPION surfaces which were partially activated through the interaction with X-rays and thereupon, were enabled to release electrons to nearby NO+ cations. MCF-7 cells containing NOBF4 stabilized SPIONs and Au-Fe3O4 NHDs and exposed to X-radiation exhibited a rise of the NO level by 60 % (AuFe3O4 NHDs) and 20 % (SPIONs) (Figure 4a). The difference in the NO production is explained with the more efficient reduction potential of X-ray activated AuNP surface in comparison with that of the SPION surface. Under X-ray irradiation Au-Fe3O4 NHDs were observed to enhance the production of peroxynitrite in MCF-7 cells by ca. 230 % which is significantly larger than the increase of the peroxynitrite level (ca. 40 %) due to the interaction of X-rays with the SPIONs (Figure 4b). This is understood to arise from both, the physical and ACS Paragon Plus Environment

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chemical impact of X-rays on AuNP surfaces as resulting in the emission of secondary electrons as well as in the creation of highly catalytically active surfaces.15 While secondary electrons predominantly boost the formation of O2-, the electron-spill out at hot spots of the strongly curved AuNP surfaces also provides the production of NO radicals. In order to gain complementary information of the X-ray induced formation of NO and peroxynitrite in the MCF7 cells vitamin B12 and uric acid, as acting as specific NO and peroxynitrite scavenger, respectively, were utilized.19,36-38 This implies that the MCF-7 cells were exposed to Au-Fe3O4 NHDs, either combined with vitamin B12 or combined with uric acid (Figure 4). As being expected, the addition of vitamin B12 led to the reduction of the NO level, whereas a similar significant effect on peroxynitrite by vitamin B12 was not observed (Figure 4). Scavenging of NO by vitamin B12 is expected to impede the formation of peroxynitrite. However, the rapid reaction of the NO radical with O2- on the nanoparticle surface outperforms the NO scavenging by vitamin B12. On the other hand, uric acid, as being a major oxidant in human plasma39, was found to selectively scavenge peroxynitrite, but did not affect the NO level in MCF-7 cells under X-ray treatment (Figure 4). Both scavengers increase the cell viability of MCF-7 cells incubated with NOBF4-stabilized Au-Fe3O4 NHDs ((SI): Figure S10)

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A uN P

increase of fluorescence A u(% of control) Fe 3O N 4 A H N uD H Fe s D + s 3O ur 4 ic N H ac D id s + vi t. B 12 SP IO N 200

increase of fluorescence A u(% of control) Fe 3O 3O 4 N 4 A H N uD H Fe s D + s 3O u 4 ric N H ac D id s + vi t. B 12 SP IO N

(b)

(a)

A uFe

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



Figure 4. Relative concentrations of the NO radical (a) and peroxynitrite (b) in MCF-7 cells incubated with NOBF4 stabilized SPIONs or Au-Fe3O4 NHDs after X-ray irradiation with 1 Gy or left non-irradiated. Cellular Defenses against Oxidative Stress. Exposure of cancer cells to clinically relevant doses of X-radiation does not only elicit DNA damage and radiolysis of water, but also may disrupt mitochondrial functions and thereupon, increases the mitochondrial oxidative stress.40 Mitochondria are the major source of O2- that is involved in the formation of peroxynitrite. Superoxide dismutases (SODs) eliminate O2- by catalyzing the conversion of O2- to O2 and H2O2 and inhibit the reaction of O2- with NO to peroxynitrite.41-42 Hence, the catalytic activity of SODs is expected to be increased in X-ray treated cells due to both, the rise of the mitochondrial generation of O2•- and the O2•- formation by X-ray induced water radiolysis. X-ray induced increase of the SOD activity was observed for the control MCF-7 cells (Figure 5a, medium). On the other hand, incubation of the MCF-7 cells, either with Au-Fe3O4NHDs or with Au-Fe3O4-NHDs plus uric acid, apparently diminishes the SOD activity before Xray irradiation. This is in line with the results presented in Figure 3b: intracellular Au-Fe3O4NHDs exhibit partially positively charged surfaces that may adsorb O2- and thereupon, hamper the SOD activity. X-ray treatment of these loaded MCF-cells resulted in a further degrading of the SOD activity. X-ray activated Fe3O4 surfaces may react with the NO+ cations through their Fe2+ ions to yield NO radicals which scavenge O2- by the formation of peroxynitrite (Eq.(3)). Moreover, peroxynitrite can inactivate SODs due to nitration of the critical tyrosine-34 residue.19 Intracellular uric acid does not affect the O2- level, but selectively scavenges peroxynitrite. Therefore, the combination of Au-Fe3O4 NHDs with uric acid gave rise to (slightly) increased SOD activities in X-ray-irradiated and non-irradiated MCF-7 cells. The cellular defense system employs, in addition to antioxidant enzymes, low molecular weight antioxidant compounds for eliminating ROS. The key survival antioxidant of cells is glutathione (GSH) which has been shown to directly scavenge a wide variety of highly reactive oxidants. In this redox reaction GSH transfers one electron to a radical and dimerizes with another oxidized GSH to glutathione disulfide (GSSG). The GSSG/2GSH couple maintains the redox homeostasis in the cell. X-ray irradiation of cells raises the production of ROS and thereupon, reduces the intracellular GSH/GSSG ratio which was observed for the control MCF7 cells (Figure 5b, medium). Intracellular Au-Fe3O4 NHDs, when interacting with X-radiation, were shown to significantly increase the formation of reactive species of oxygen and nitrogen (Figures 3 and 4). As an immediate reaction of the intracellular defense system the GSH/GSSG ratio considerably fell off (Figure 5b). The strong oxidant peroxynitrite reacts with low-molecular weight and protein-bound thiols. Hence GSH provides a major protection mechanism against ONOO- dependent oxidative damage.20 Due to selectively scavenging ACS Paragon Plus Environment

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peroxynitrite intracellular uric acid resulted into a considerably higher GSH/GSSG ratio in both, X-ray irradiated and non-irradiated MCF-7 cells (Figure 5b).

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20 15 10 5

ratio GSH to GSSG

SOD activity (units/mg protein)

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m iu ed

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ic ur

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id

Figure 5. The SOD activity (a) and the GSH/GSSG ratio (b) in Au-Fe3O4 NHDs loaded MCF7 cells either with or without uric acid before and after X-ray irradiation.

Impact of the RONS on the Membrane Lipid Peroxidation, DNA Fragmentation and Cell Death. Peroxynitrite, when generated in excess, may defeat the antioxidant defense system and thereupon, may initiate membrane lipid peroxidation (MLP). ONOO- degrades lipid molecules directly or through its highly reactive products NO and OH emerging from its proton-catalyzed decomposition. The reaction between these different kinds of radicals and the unsaturated fatty acids yields a broad variety of oxidation and nitration products. Several nitrogen-containing lipid intermediates may decompose to highly reactive radicals. In case of a sufficiently high radical concentration a self-sustaining peroxidation reaction will be initiated.21 Malondialdehyde (MDA) being a convenient biomarker for lipid peroxidation was utilized as a quantitative probe for MLP. X-ray irradiation of MCF-7 cells incubated with AuFe3O4 NHDs was found to cause the formation of peroxynitrite and thereupon, gave rise to membrane lipid peroxidation. This is demonstrated by the 200 % increase of the malondialdehyde level in MCF-7 incubated with Au-Fe3O4 NHDs (Figure 6a). The impact of Au-Fe3O4 NHDs on the peroxynitrite generation in X-ray treated MCF-7 cells was apparently reduced by uric acid (Figure 6b). Peroxonitrite-induced MLP is immediately associated with mitochondrial outer membrane permeabilization (MOMP) which is a key feature of peroxynitrite-mediated



apoptosis.15 MOMP facilitates the efflux of various pro-apoptotic signaling molecules which promote apoptosis. A peroxynitrite stimulated release of the apoptosis-inducing factor from the mitochondria triggers a DNA fragmentation process. This mitochondrial process is accompanied by a nuclear process, in which peroxynitrite directly induces DNA strand breaks.19-21 Thus, X-ray-induced peroxynitrite formation should lead to an increase in DNA fragmentation inside the nucleus. This was verified using the TUNEL assay, which selectively labels the 3’-OH termini of DNA strand breaks (Figure 6b). X-ray irradiation of MCF-7 cells loaded with Au-Fe3O4 NHDs was found to increase the fraction of TUNEL positive cells for 44 %, whereas the addition of uric acid decreased this fraction to 24 %. Evidently the impact of X-rays on Au-Fe3O4 NHDs internalized in MCF-7 cells gives rise to the formation of peroxynitrite at a sufficiently high concentration which causes membrane lipid peroxidation as well as induces DNA fragmentation which is expected to introduce apoptosis. In a complementary study, by employing the clonogenic cell survival assay, we examined the influence of Au-Fe3O4 NHDs and SPIONs on the survival and proliferation of the MCF-7 cells exposed to X-radiation at the single doses 1 Gy, 2 Gy and 3 Gy (Figure 6c). As expected, the survival curves of X-ray irradiated MCF-7 cells loaded with the diverse nanoparticles exhibited a significantly larger decrease than that of the control cells (medium). The X-ray-induced catalytic activity of the SPIONs was reduced because of surface oxidation. Fe2O3 surfaces are rather inefficient for the production of secondary RONS which explains the smaller decrease of the survival curve in comparison of those obtained for the Au-Fe3O4 NHD loaded MCF-7 cells. Hence, the survival curves confirm that the NOBF4-stabilized Au-Fe3O4 NHDs perform as highly efficient X-ray enhancer. This is corroborated by the corresponding dose modifying factor. The NOBF4-stabilized Au-Fe3O4 NHDs exhibit a DMF value with 0.366 which is significantly smaller than that of the SPIONs (0.783) and that of the AuNPs (0.616).


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mol MDA/mg protein

(a)


0.4 0.3 0.2 0.1 0.0

M

iu ed

m O e3 F uA

4

H N

O e3 F uA

s D

4

H N

s D

+

ic ur

(b) non-irradiated irradiated

50 40 30 20 10 0

id ac

M

m iu d e

3O Fe u A

u A

H N 4

O e3 -F

s D s D H N -

+

ic ur

i ac

d

4

(c)

surviving fraction

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


TUNEL-positive cells (%)

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Medium SPION Au-Fe3O4 NHDs

1.0

AuNPs 0.5

0.0 0

1

2

3

4

radiation dose [Gy] Figure 6. X-ray-induced membrane lipid oxidation in MCF-7 cells in medium or incubated with only Au-Fe3O4 NHDs or in combination with uric acid and exposed to a 1 Gy dose which raises the malondialdehyde (MDA) level (a), X-ray- induced DNA fragmentation, which is visualized by TUNEL assay (b); cell survival curves of MCF-7 cells loaded with SPIONs, AuNPs and AuFe3O4 NHDs under X-ray exposure at dosages of 1 Gy, 2 Gy and 3 Gy (c).

CONCLUSIONS Snowman-shaped oleic acid/oleylamine terminated Au-Fe3O4 NHDs with sizes around 11 nm were successfully synthesized through thermal decomposition of Fe(III)oleate and subsequent epitaxial growing of Fe3O4 on 6 nm-sized Au seeds at 320°C. A partial ligand exchange with ACS Paragon Plus Environment


NOBF4 was achieved through a two-phase reaction, and water-soluble NO+ functionalized AuFe3O4 NHDs were obtained. The morphologic and structural properties and composition of the Au-Fe3O4 NHDs were verified using TEM and XRD. The crystalline SPION and gold components were shown to exhibit superparamagnetism and surface plasmon resonance, respectively. The red-shift and broadening of the surface plasmon resonance band in comparison with equally-sized AuNPs indicate efficient electron transfer across the Au-Fe3O4 interface which evidently augments the catalytic activity of the Fe3O4 surface. Good water dispersibility of the NOBF4 stabilized Au-Fe3O4 NHDs was verified by DLS measurements. Biocompatibility of Au-Fe3O4 NHDs or SPIONs was examined and proven by performing the neutral red cytotoxicity assay on MCF-7 cells incubated with these nanoparticles. The impact of X-radiation on NOBF4-stabilized SPIONs and Au-Fe3O4 NHDs, as consisting in Fe3O4surface mediated electron transfer to the NO+ cations, was shown to create NO radicals. Moreover, we could establish a synergetic interplay between the X-ray-irradiated Au and Fe3O4 surfaces of intracellular Au-Fe3O4 NHDs which results into the simultaneous production of the NO radicals at the Fe3O4 surface and the O2- radicals at the Au surface. In a surface-confined reaction the NO and O2- radicals efficiently form peroxynitrite. This highly reactive radical species may cause nitration of mitochondrial proteins, lipid peroxidation, and induces DNA fragmentation either by the release of mitochondrial apoptosis-inducing factor or by direct DNA oxidation. This destructive effectiveness of peroxynitrite and other ROS, originating from X-ray exposure of intracellular Au-Fe3O4 NHDs, on the membrane lipids, viability and proliferation of MCF-7 cells, was verified by detecting a respectively increased level of MDA and DNA fragmentation as well as considerably reduced survival fractions. In a nutshell, NOBF4stabilized Au-Fe3O4 NHDs were demonstrated to provide a powerful nanoplatform for X-ray induced formation of the highly reactive radicals NO, the O2- and ONOO- and therefore, exhibit an excellent performance as X-ray enhancing agents in radiation therapy.

EXPERIMENTAL SECTIONS 2.1 Chemicals: Tetrachloroauric(III)acid trihydrate (HAuCl4*3H2O, ≥99.5 %), ethanol (≥99.8%) and methanol (≥99.8%) were purchased from Carl Roth GmbH + Co. KG. Ammonium iron(II) sulfate hexahydrate ((NH4)2Fe(SO4)2*6H2O, ≥99%), iron(III) chloride hexahydrate (FeCl3*6H2O, 97%), oleic acid (90%), 1-octadecene (90%), tert-butylamine borane (TBAB, 97%), nitrosyl tetrafluoroborate (NOBF4, 95%), chloroform (≥99.9%), toluene (≥99.5%), iso-propanol (≥98%), sodium hydroxide (NaOH, 97%), ammonia solution (NH4OH, 30 wt.%), penicillin-streptomycinsolution, sodium pyruvate, phosphate buffered saline (PBS), non-essential amino acids (MEM), trypsin/EDTA, 3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride (neutral red), bovine serum albumin (BSA, 95 %), Tris-HCl (99 %), di-sodium EDTA (99 %), D-(+)ACS Paragon Plus Environment

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Glucose, HEPES sodium salt (99 %), MgSO4 anhydrous (≥ 99,5 %), trichloroacetic acid (TCA) (99 %), 2-thiobarbituric acid (TBA) (98 %), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) (98 %), 2-vinylpyridine (97 %), triethanolamine (99 %), NADPH (≥ 97 %), glutathione reductase, 5’sulfosalicylic acid (≥ 99 %), bicinchoninic acid protein assay kit, reduced and oxidized glutathione (98 %), pyrogallol (98 %), Hoechst 33258 solution (1mg/mL), crystal violet (98%) and 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (95 %) were purchased from Sigma-Aldrich Co. LLC. 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide was obtained from Sikémia. DMEM, fetal calf serum (FCS), GlutaMAX Supplement, MitoSOXTM Red Mitochondrial Superoxide Indicator and 3-(p-hydroxyphenyl) fluorescein (HPF) were bought from Thermo Fischer Scientific and glacial acetic acid, acetonitrile, NaHCO3 (99.5%), CaCl2 anhydrous (p. a.), KH2PO4 (p. a.) and K2HPO4 (99 %) and DMSO (99.7%) from Merck. Oleylamine (80-90%) was purchased from Acros Organics, n-hexane from VWR, Triton X-100 from Riedel de Haën and NaCl (99.5%), KCl (99.5%) and MgCl2 anhydrous (98 %) were obtained from Fluka. The malondialdehyde standard, DAN-1EE hydrochloride and dihydrorhodamine 123 (DHR) was ordered from Cayman Chemical Company, and the in-situ Cell Death Detection Kit, TMR red was purchased from Roche Applied Science. Ultrapure water with a conductivity of 18 MΩcm-1 was used during the experiments. 2.2 Synthesis and Preparation of the Nanoparticles 2.2.1 Iron(III) oleate (Fe(OL)3) precursor. The iron oleate precursor was synthesized following Kovalenko’s procedure.29 3.25 g FeCl36H2O (12 mmol) were dissolved in 30.0 mL methanol and combined with 12.7 mL oleic acid (40.0 mmol). A 0.4 M solution of NaOH (36.0 mmol) was dropwisely added to the reaction mixture under vigorous stirring, until a brown solid was precipitating. The obtained brown precipitate was washed twice with methanol and was dissolved in n-hexane. This solution was further washed thrice with warm Milli-Q water (50°C) using a separatory funnel. The solvent was removed under reduced pressure, and a brown waxy product was obtained. 2.2.2 Gold nanoparticles in organic solvent. Gold nanoparticles (AuNPs) were synthesized through the reduction of HAuCl4.3H2O with TBAB in the presence of chloroform and oleylamine.43 40.0 mg HAuCl4.3H2O (0.102 mmol) were dissolved in 4.00 mL chloroform and further 4.00 mL of oleylamine (12.2 mmol) were added. The precursor solution was stirred for 10 min in air at 0°C. 35.0 mg tert-butylamine borane (0.402 mmol) and 400 µL oleylamine (1.22 mmol) in 400 µL chloroform were mixed by sonication and added to the precursor solution. The color of the reaction mixture gradually changed from orange into purple. The mixture was stirred for one hour at 0°C before the NPs were precipitated by the addition of ethanol (30 mL). The AuNPs were collected by centrifugation (8500 rpm, 10 minutes) and were dispersed in n-hexane. After further washing with ethanol (30 mL) (8500 rpm, 10 min) they were stored in n-hexane (10 mL).



2.2.3 Snowman-shaped Au-Fe3O4 NHDs. The snowman-shaped Au-Fe3O4 NHDs were prepared following the thermal decomposition procedure of Lee et al.35 Under argon atmosphere the pre-synthesized Au seeds in n-hexane were added to a solution containing 15.0 mL 1-octadecene, 1.00 mL oleic acid (3.15 mmol) and 1.00 mL oleylamine (3.04 mmol). The reaction mixture was stirred for 30 min at 120°C. Then Fe(III)oleate (Fe(OL)3) (230 mg, 0.250 mmol) was dissolved in 5.00 mL 1-octadecene and injected into the hot solution. The reaction mixture was heated to 320°C and kept at this temperature for 4 h. After cooling down to room temperature the reaction mixture was exposed to air for 1 h before iso-propanol (60 mL) was added. The Au-Fe3O4 NHDs were collected by centrifugation (8500 rpm, 10 min), re-dispersed in n-hexane and washed with ethanol (40 mL) (8500 rpm, 10 min). Finally, the NHDs were stored in n-hexane (10 mL). 2.2.4 Surface-coating with NOBF4. The Au-Fe3O4 NHDs were made water soluble by a phase transfer reaction.31 75.3 mg of the NHDs were dissolved in 15.1 mL n-hexane and then mixed with a solution consisting of 88.0 mg NOBF4 (0.753 mmol) in 15.1 mL acetonitrile. After 2 h, the NHDs were transferred from the upper hexane layer to the bottom acetonitrile layer. The NHDs were precipitated by the addition of toluene (40 mL) and collected by centrifugation (10000 rpm, 10 min). Finally the NHDs could be dispersed in Milli-Q water. 2.3 Instrumentation. Transmission electron microscopy (TEM) images were recorded using a Zeiss EM 900 transmission electron microscope (Carl Zeiss, Oberkochen, Germany). The magnetic behavior of the NHDs was investigated with a Quantum Design MPMS-XL5 superconducting quantum interference device (SQUID) magnetometer (Qantum Design, San Diego, California, USA). Fourier transform infrared spectra were recorded on a Bruker Tensor 27 spectrometer (Bruker, Billerica, Massachusetts, USA). XRD measurements were performed using a Philips/PANalytical X-Pert device (Philips/PANalytical, Almelo, Netherlands) that operates with Ni-filtered copper radiation. Dynamic Light Scattering (DLS) NHDs size distribution and zeta potential were obtained by measuring a 0.2 wt % dispersion of NHDs in hexane or PBS. The cell viability and various radical assays were measured using a Synergy HT microplate reader (BioTek Inc.,USA) The TEM images of the MCF-7 cells loaded with the different nanoparticles were collected using a Zeiss EM 906 (Germany). The cells were exposed to X-radiation using a 120 kV X-ray tube equipped with a tungsten anode adjusted to a mean energy of 34 keV and maximum energy of 120 keV (Comet MXR 160/0.4-3.0, Switzerland). 2.4 Cell culture. Human breast adenocarcinoma cells (MCF-7 cells) were cultured in DMEM containing 4500 mg glucose/L and being supplemented 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. The MCF-7 cells were stored at 37°C in a humidified 5 % CO2 atmosphere and were sub-cultivated twice a week.


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2.5 ROS Detection Assays. The MCF-7 cells were seeded in 96 well plates at a density of 23104 cells per well and were incubated overnight. After the removal of the cell medium solutions containing nanoparticles at the concentration of 10 μg/mL in medium were added into the wells, and the plates were placed in the incubator overnight. The nanoparticle solutions were aspirated off. According to the respective assay protocol (see supporting information (SI)) the respective dye solution was filled into the wells. One half of the plates were exposed to Xrays with a single dose of 1 Gy. 2.6 RNS Detection Assays. The MCF-7 cells were seeded in 96 well plates at a density of 23104 cells per well and incubated overnight. The cell medium in the wells was substituted by the cell medium solution containing NOBF4-stabilized Au-Fe3O4 NHDs at a concentration of 10 μg/mL. A solution containing the peroxynitrite scavenger uric acid at a concentration of 0.2 mM was added to 48 wells, whereas the other 48 wells were supplied with the NO scavenger vitamin B12 (0.5 mM). The plates were placed in the incubator overnight. The solutions were aspirated off. The respective assay uses a DAN-1EE dye solution for the measurement of the nitric oxide radical and a dihydrorhodamine 123 dye solution for the peroxynitrite measurement. Therefor the respective dye solution was filled into the wells of the plates. Half of the plates were exposed to X-rays with a single dose of 1 Gy. 2.7 Preparation of the cell extract. The cells were seeded into 6-well plates at a density of 3105 per well and incubated overnight for attachment. Afterwards the cells were treated with 10 µg/mL of the Au-Fe3O4 NHDs in cell medium. One well was supplied with the peroxynitrite scavenger uric acid (0.2 mM) and another one with the NO scavenger vitamin B12 (0.5 mM). One half of the 6-well plates were irradiated with X-rays with a single dose of 1 Gy and the other one was left non-irradiated. After 6 h X-ray irradiation the cell medium was aspirated off and the cells were lysed in cell lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 1 % Triton-X 100. For the measurement of the GSH level 0.6 % sulfosalicylic acid was added. The cell lysate was homogenized on ice by sonication. After centrifugation (15000 g, 10 min, 4°C) the cell extract (supernatant) was stored on ice. 2.8 Measurement of the protein concentration. The data were normalized by determining the protein concentration. Therefor the protein concentration was measured by performing the BCA Protein Assay Kit according to the manufacturer´s instruction. The calibration curve was obtained using BSA as the standard. 2.9 Superoxide-dismutase (SOD) activity assay. The SOD activity was examined following the procedure by Marklund and Marklund44 which consists in measuring the inhibition of the autoxidation of pyrogallol. The assay mixture contained 50 mM Tris-HCl (pH 8.2), 1 mM EDTA and 0.04 mM pyrogallol (stock solution: 0.4 mM dissolved in 0.01 N HCl). The reaction was initiated by the addition of the enzyme sample or cell extract. The absorption at 325 nm is



measured every 30 s for 5 min at 37°C. One unit of enzyme was defined as amount of SOD required to produce half of the maximum inhibition of the autoxidation. 2.10 Ratio of reduced and oxidized glutathione (GSH/GSSG). This assay is composed of two parts: the detection of the GSSG concentration and of the total concentration of GSH and GSSG. Upon following Griffith´s method for the GSSG assay, 2 µL 2-vinylpyridine (diluted 1:10 in potassium phosphate (KP) buffer (pH 7.4)) is added to 100 µl cell extract. After 1 h surplus 2-vinylpyridine is neutralized using 6 µL triethanolamine (diluted 1:6 in KP buffer). For the determination of the total GSH concentration the cell extract is used as it is. 20 µL of the cell extract or the cell extract containing 2-vinylpyridine is mixed with 20 µL β-NADPH solution (2 mg in 3 mL KP buffer) and 40 µL 5,5’-dithio-bis(2-nitrobenzoic acid (DNTB)-glutathione reductase (GR) solution (2 mg DTNB, 40 µL (250 units/mL) GR, in 3 mL KP buffer). The absorbance at 412 nm was measured every 30 sec. for 2 min. The GSH concentration was calculated using the relationship [GSH]total = [GSH] + 2  [GSSG].45 2.11 Malondialdehyde (MDA) level. The level of membrane lipid peroxidation (LPO) was determined by measuring the formation of malondialdehyde (MDA) as a product of membrane LPO.46 A mixture of 0.1 mL cell extract, 0.75 mL 5 % trichloroacetic acid (TCA) and 0.75 mL 1 % thiobarbituric acid (TBA) (dissolved in 1 M KOH) was heated at 90°C for 30 min. After cooling down to room temperature the mixture was centrifuged (2300 g, 15 min) in order to collect the supernatant. The fluorescence emission of the supernatant was excited at 532 nm excitation and detected at 553 nm. The concentration of MDA in μmole/mg protein was calculated with the help of a calibration curve using a MDA standard. 2.12 TUNEL assay. The MCF-7 cells were seeded on cover slides and incubated overnight. Afterwards the medium was replaced by one containing the Au-Fe3O4 NHDs or the NHDs in combination with either uric acid or vitamin B12. The TUNEL assay was performed according to the manufacturer’s instructions. Afterwards the cell nuclei of all cells were stained with Hoechst 33258 (1 μg/mL in PBS) to determine the ratio of apoptotic cells. 2.13 Clonogenic cell survival assay. This assay was described in detail in ref.47. The MCF7 cells were grown in 6-well plates and incubated overnight with the diverse nanoparticles at a concentration of 10 µg/mL. After X-ray irradiation between 0 and 3 Gy the cells were detached, seeded and grown in 6-well plates for 2 weeks to form colonies. The colonies were fixed and stained with a mixture of 0.5 % (w/v) crystal violet in 50/50 methanol/water for 30 min. The count of colonies containing more than 50 cells was used for the calculation of the surviving fraction (SF). The survival curves were fitted to a linear quadratic function (ln SF = -(αD+βD2)). In order to quantify the X-ray enhancing effect the dose modifying factor (DMF) was calculated from the X-radiation survival curves by taking the ratio of radiation doses at the 50 % survival level (nanoparticle-induced radiation dose divided by the control radiation dose). DMF values smaller than 1 indicate an X-ray enhancing effect.


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Associated content Supporting information Additional information for the Experimental Section; the size distribution of the Au-Fe3O4 NHDs according to the TEM images, UV/Vis absorption spectra of AuNPs and the Au-Fe3O4 NHDs, images of the oleic acid/oleylamine coated Au-Fe3O4 NHDs in hexane and their transfer to the acetonitrile phase during the NOBF4 stabilization, FT-IR spectrum Au-Fe3O4 NHDs coated with NOBF4 in comparison with oleic acid and oleylamine, the magnetization curve of NOBF4 stabilized Au-Fe3O4 NHDs, cell viability assays, TEM image of MCF.7 cells loaded Au-Fe3O4 NHDs, change in the relative ROS, superoxide and hydroxyl radical concentration in MCF-7 cells incubated with AuNPs, results of the HPF assay for the Au-Fe3O4 NHDs and SPIONs

Author information Corresponding author Carola Kryschi *E-mail: [email protected] ORCID: 0000-0003-4939-5886 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements We thank Andrea Hilpert for TEM studies (Department of Anatomy, Chair of Anatomy I, University of Erlangen) and Tobias Weißenberger for the ICP-OES measurements. (Prof. Dr. Peter Wasserscheid, Institute of Chemical Reaction Technique, University of Erlangen).

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150x125mm (300 x 300 DPI)


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Functionalized Au-Fe

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