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Magnetic Reactive Oxygen Species Nanoreactor for Switchable Magnetic Resonance Imaging Guided Cancer Therapy Based on pH-Sensitive Fe5C2@Fe3O4 Nanoparticles Jing Yu, Fan Zhao, Weiliang Gao, Xue Yang, Yanmin Ju, Lingyun Zhao, Weisheng Guo, Jun Xie, Xing-Jie Liang, Xinyong Tao, Juan Li, Yao Ying, Wangchang Li, Jingwu Zheng, Liang Qiao, Subin Xiong, Xiaozhou Mou, Shenglei Che, and Yanglong Hou ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b01740 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Magnetic Reactive Oxygen Species Nanoreactor for Switchable Magnetic Resonance Imaging Guided Cancer

Therapy

Based

on

pH-Sensitive

Fe5C2@Fe3O4 Nanoparticles Jing Yu,1, ¶ Fan Zhao, 1, ¶ Weiliang Gao,1 Xue Yang,2 Yanmin Ju,3 Lingyun Zhao,4 Weisheng Guo,5 Jun Xie,6 Xing-jie Liang,5 Xinyong Tao,1 Juan Li,1 Yao Ying,1 Wangchang Li,1 Jingwu Zheng,1 Liang Qiao,1 Subin Xiong,7 Xiaozhou Mou,2,* Shenglei Che,1,* and Yanglong Hou3,* 1College

of Materials Science and Engineering, Zhejiang University of Technology, Hangzhou

310014, China 2Clinical

Research Institute, Zhejiang Provincial People’s Hospital, Hangzhou 310014, China

3Beijing

Key Laboratory for Magnetoelectric Materials and Device, Beijing Innovation Center

for Engineering Science and Advanced Technology, Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China 4Key

Laboratory of Advanced Materials, Ministry of Education, School of Material Science &

Engineering, Tsinghua University, Beijing 100084, China

ACS Paragon Plus Environment

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Center for Excellence in Nanoscience, Chinese Academy of Sciences, CAS Key

Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190, China 6School

of Life Science, Jiangsu Normal University, Xuzhou 221116, China

7College

of pharmacy, Zhejiang University of Technology, Hangzhou 310014, China

*Corresponding

authors

(E-mail:

[email protected];

[email protected];

[email protected])

Keywords iron carbide nanoparticles, pH sensitive, reactive oxygen species, switchable magnetic resonance imaging, cancer, imaging guided therapy

Abstract

Reactive oxygen species (ROS) are crucial molecules in cancer therapy. Unfortunately, the therapeutic efficiency of ROS is unsatisfactory in clinic, primarily due to their rigorous produced condition. By taking advantage of intrinsic acidity and overproduction of H2O2 in the tumor environment, we have reported an ROS nanoreactor based on core-shell-structured iron carbide (Fe5C2@Fe3O4) nanoparticles (NPs), through the catalysis of Fenton reaction. These NPs are able to release ferrous ions in acidic environments to disproportionate H2O2 into ·OH radicals,


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which effectively inhibits the proliferation of tumor cells both in vitro and in vivo. The high magnetization of Fe5C2@Fe3O4 NPs is favorable for both magnetic targeting and T2-weighted magnetic resonance imaging (MRI). Ionization of these NPs simultaneously decreases the T2 signal and enhances the T1 signal in MRI, and this T2/T1 switching process provides the visualization of ferrous ions release and ROS generation for the supervision of tumor curing. These Fe5C2@Fe3O4 NPs show great potential in endogenous environment-excited cancer therapy with high efficiency and tumor specificity, and can be guided further by MRI.

Reactive oxygen species (ROS) are chemically reactive molecules, that are essential for the cell life cycle at low concentrations, but induce cell apoptosis or necrosis at concentrations above a certain threshold.1-3 ROS-mediated cancer treatment based on generation of ROS in situ has gained widespread attentions in recent years.4-6 Photodynamic therapy (PDT) is the most typical strategy for generating ROS, via an interaction among light, a photosensitizer, and oxygen.7-11 However, the clinical application of PDT is hindered by low light penetration, photosensitizer self-catalysis, or its reliance on oxygen and tumor anoxia.12,13 Fenton reaction is an old but promising reaction that efficiently generates a specific kind of ROS, hydroxyl radical, by the disproportionation of hydrogen peroxide (H2O2) with Fe2+ ions.14 Benefiting from the accumulation of H2O2 in the tumor environment, it can overcome the limitations of PDT, and directly induce cell death without external energy, it has been applied extensively in ROS-related cancer therapy.15-18 Ferrous ions are essential for ROS generation in Fenton reaction. However, when free Fe2+ ions are injected into the body, few could reach the target tumor for ROS production, owing to


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their wide biodistribution throughout the whole body. Nonspecific oxidation in blood circulation, which is caused by the low potential of the Fe2+/Fe3+ redox couple (0.77 eV), results in serious side effects when other organs overproduce H2O2.19,20 Developing materials that specifically release Fe2+ at the tumor site is imperative. Fortunately, in addition to overproducing peroxide, cancerous regions are also characterized by acidity.21 Thus, iron-based materials with pHsensitivity can satisfy the demand. Currently, the most widely applied pH-responsive iron-based materials are Fe3O4 nanoparticles (NPs).22,23 Fe3O4 NPs can induce tumor cell apoptosis through ROS generation,24-26 and also can be applied in MRI as T2 contrast agents.27-29 Recently, amorphous iron NPs were reported to be more sensitive to acidity, and to burst release Fe2+ ions even in extracellular tumor environments, thus showing great promise in ROS-related cancer therapy.19,30,31 Iron carbide nanoparticles (ICNPs) are nanointermetallic compounds that consist of iron and carbon, and might be good candidates for ferrous ion-dependent therapy.32 In comparison with metal iron NPs, ICNPs have carbon atoms that occupy interstitial sites of the iron lattice, giving these NPs higher stability when they are exposed to an oxygen/water-containing atmosphere, which may improve their biosafety and useful lifespan.33,34 Their high saturation magnetization (~140 emu g-1) further expands their applications in biomedicine and magnetic storage.35-38 In our previous study, a typical configuration of ICNPs (Fe5C2) with a core shell structure was applied in cancer theranostics, such as image-guided therapy and stimuli controlled therapy,39,40 and the carbon shell coating on these ICNPs maintained a long-lasting high magnetic property for better magnetic resonance imaging (MRI).41 However, this carbon protection makes ICNPs inert to their surroundings, and leads to the unreliability of pH-controlled iron ion release. Considering the fact that ICNPs are intrinsically intermetallic compounds, in which the valence


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state of iron tends to be zero, these NPs should be sensitive to an acidic environment. Therefore, we decided to develop another kind of ICNP for tumor environment-targeted ferrous ion delivery with high stability in physiological environments. Herein, we fabricated a core-shell structured iron-based NPs with a magnetic Fe5C2 core and pH-responsive iron oxide coating on the shell (Fe5C2@Fe3O4 NPs), which efficiently released the H2O2 “catalyst” ferrous ions in acidic environments and exhibited high magnetization in normal tissue. In comparison with traditional ferrous ions donors, iron oxide (Fe3O4) NPs, Fe5C2@Fe3O4 NPs are more sensitive to acidity, and can discharge ferrous ions more effectively in low-pH environments. Combined with the overproduction of H2O2 in tumor regions, the designed Fe5C2@Fe3O4 NPs are able to catalyze a higher level of ROS generation, which results in more tumor-selective therapy with low toxicity. Benefiting from the high magnetic property, an enrichment of NPs and “burst” release of iron ions could be produced by locating a magnet at the tumor site, and this magnetic targeting process could be monitored through T2-weighted MRI. More interestingly, the efficient discharge of iron ions from Fe5C2@Fe3O4 NPs at the tumor site decreased the magnetism of the NPs, which reduced the T2 signal in the MRI, and at the same time, released T1 “signal enhancer” ferrous ions for lightening on T1-weighted MRI. This T2/T1 signal conversion sensitively reported the production of ferrous ions and, consequently, traced the release of ROS for visible cancer therapy. Compared with other commonly applied monitorable therapeutic agents, in which the imaging components and therapeutic parts are usually separate constituents, these Fe5C2@Fe3O4 NPs are able to produce a diagnostic signal and curing effect with the same constituent, thus making real imaging-traceable therapy feasible. Our result shows the potential of Fe5C2@Fe3O4 NPs in spontaneous, tumor-selective, and image-


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guided therapy based on ROS generation and switchable MRI without external stimuli (Figure 1).

Figure 1. A schematic illustration of Fe5C2@Fe3O4 NPs for pH-responsive Fe2+ releasing, ROS generation and T2/T1 signal conversion.


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Results and Discussion Synthesis and Characterization of Fe5C2@Fe3O4 NPs Fe5C2@Fe3O4 NPs were synthesized by a two-step method following our previously reported process with a slight modification.33 Representative transmission electron microscopy (TEM) images of the as-synthesized NPs revealed that the NPs are in a core/shell structure with an overall size of approximately 20 nm, which was consistent with the dynamic light scattering (DLS) data (Figure 2a and Figure S1). The core size of Fe5C2@Fe3O4 NPs is measured to be 16 ± 2 nm, while the shell thickness is approximately 2 ± 1 nm. High-resolution TEM (HRTEM) showed that the core part was highly crystallized, with the chemical structure of Fe5C2; the shell region was composed of amorphous entities with several tiny crystalline domains, which represented the iron oxide Fe3O4 with an inverse spinel structure (Figure 2b).33 Observations from X-ray powder diffraction (XRD) analysis confirmed that the phase in the core is Fe5C2, while no obvious peak from Fe3O4 was observed, due to its low crystallization (Figure 2c). To verify the composition of the amorphous shell, X-ray photoelectron spectroscopy (XPS) was applied, indicating the coexistence of carbon and iron (Figure 2d). Two peaks in the Fe 2p spectrum, at ~710 and ~724 eV, can be assigned to Fe3O4, and the results fit well with HRTEM (Figure S2).42 Interestingly, a void space was clearly observed between the Fe5C2 core and Fe3O4 shell (Figure 2a and Figure 2b), this may be the active area for some chemical reactions such as the carbon penetration process,33,43 endowing the chemical activity of Fe5C2@Fe3O4 NPs. The NPs were relatively stable in air, with their morphology and size distribution unchanged within 7 days (Figure S3 and Figure S4).


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Figure 2. Characterization of Fe5C2@Fe3O4 NPs. (a) TEM image of 20 nm Fe5C2@Fe3O4 NPs. (b) HRTEM image of a single Fe5C2@Fe3O4 NPs. (c) XRD pattern of Fe5C2@Fe3O4 NPs (JCPDS no. 36-1248, 76-1849). (d) XPS survey spectrum of Fe5C2@Fe3O4 NPs.

pH-Dependent PEG/Fe5C2@Fe3O4 NP Decomposition and Fe2+ Release Behaviors Compared with carbon-shelled Fe5C2 (Fe5C2@C) NPs of similar size obtained by our previously reported one-step synthesis (Figure S5),42 these Fe3O4-coated Fe5C2 NPs are less stable against dissolution in acidic environments. The degradation process of polyethylene glycol (PEG) modified Fe5C2@Fe3O4 (PEG/Fe5C2@Fe3O4) NPs at pH 5.4 was monitored by TEM and DLS. The shell of PEG/Fe5C2@Fe3O4 NPs was decomposed first, followed by the resolvation of the Fe5C2 core, resulting in the collapse of NPs into small pieces within 4 h (Figure 3a, Figure


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S6 and Figure S7). The solution gradually turned bright yellow in color, suggesting the efficient release of iron ions (Figure S8). The ionizations of NPs at different pH values were investigated by ICP-AES, which suggested that a rapid release of iron ions from PEG/Fe5C2@Fe3O4 NPs in solution of pH 5.4 was achieved, with the value reaching 59.5% at 48 h (Figure 3b). In contrast, only 10% iron ions discharged from PEG coated Fe5C2@C (PEG/Fe5C2@C) NPs during 4 h under the same conditions, with their morphology remaining unchanged, and the release efficiency was raised to merely 18.9% by prolonging the incubation time to 48 h, owing to the protection by the carbon (Figure 3b and Figure S9).40 The dissociation of iron ions from PEG/Fe5C2@Fe3O4 NPs at the very beginning was similar to that from PEG protected Fe3O4 (PEG/Fe3O4) NPs; while more efficient release was achieved later, which can be attributed to the exposure of Fe5C2 after corrosion of the Fe3O4 coating. The intermetallic compound of Fe5C2 was more active in acidic environments and was able to liberate iron ions more easily. The presence of voids in Fe5C2@Fe3O4 NPs also expanded more active sites for acid etching.33 In addition, it is thought that the amorphization of nanoparticles could improve their sensitivity to pH.19,44 Thus, Fe5C2@Fe3O4 NPs with amorphous Fe3O4 shells could further raise their acidic responsivity. The results above demonstrated that PEG/Fe5C2@Fe3O4 NPs are good iron ion donors. Even under mildly acidic conditions, e.g., the extracellular tumor microenvironment (pH 6.5), up to approximately 20% of iron ions can still be leached from PEG/Fe5C2@Fe3O4 NPs, while less than 8% and 3% of NPs degraded for the PEG/Fe3O4 and PEG/Fe5C2@C groups, respectively (Figure S10). More interestingly, none of the PEG/Fe5C2@Fe3O4, PEG/Fe3O4 or PEG/Fe5C2@C NPs abundantly released iron ions under physiological environments (pH 7.4),


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and their morphology remained unchanged, suggesting their high stability for potential biomedical applications (Figure 3c and Figure 3d). Considering that ferrous ions are the key element for ROS generation, potassium ferricyanide and potassium thiocyanate were added to check the valence states of the iron ions. A blue product was observed in the reaction of PEG/Fe5C2@Fe3O4 NPs with potassium ferricyanide at pH 5.4, whereas, there was no obvious color change after incubating NPs with potassium thiocyanate, indicating the presence of ferrous ions rather than Fe3+ (Figure 3a and Figure S11). These results suggested that Fe5C2@Fe3O4 NPs were the preferable ferrous ion carrier, as they would release ferrous ions on-demand in the acidic tumor environment, but remain stable in neutral normal tissues.

Figure 3. pH-dependent PEG/Fe5C2@Fe3O4 nanoparticle decomposition and Fe2+ release behaviors. (a, c) TEM image of PEG/Fe5C2@Fe3O4 NPs by dispersing in (a) pH 5.4 and (c) pH


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7.4 solution for 4 h. Inserts are photos of potassium ferricyanide dispersed PEG/Fe5C2@Fe3O4 NPs solution. (b, d) Accumulative iron ion release of PEG/Fe5C2@Fe3O4, PEG/Fe5C2@C and PEG/Fe3O4 NPs at (b) pH 5.4 and (d) pH 7.4.

pH-Dependent MRI Model Switching of PEG/Fe5C2@Fe3O4 Nanoparticles PEG/Fe5C2@Fe3O4 NPs were magnetic materials with a high saturation magnetization value of 97 emu g-1 at room temperature (Figure S12), showing their potential as contrast agents for T2 MRI. These NPs can induce strong hypo-intensities with a transverse relaxivity (r2) of 203.83 L mmol-1 s-1 under physiological conditions, and a decreased r2 value of 64.18 L mmol-1 s-1 under the lysosome-mimic environment of pH 5.4, due to the dissolution of magnetic NPs (Figure 4a). Interestingly, the T1-weighted MR image was simultaneously lightened by exposing NPs to acid conditions, and a notable increase in longitudinal relaxivity (r1) was observed (Figure 4b), implying that the release of ferrous ions could be traced by the T2 to T1 MRI transition. Inspired by the Fenton reaction, in which Fe2+ can decompose H2O2 to ·OH, methylene blue (MB), a dye that can be degraded by ROS was applied to indicate ROS generation.45,46 A significant decrease in the absorbance was observed when MB was incubated with PEG/Fe5C2@Fe3O4 NPs and H2O2 at pH 5.4, whereas only a slight change in MB absorbance was detected after the same treatment in pH 7.4 solution (Figure 4c). This demonstrated that ROS generation was accelerated by the dispersion of PEG/Fe5C2@Fe3O4 NPs in a low-pH environment with the assistance of H2O2. Electron spin resonance (ESR) spectroscopy further confirmed that the produced ROS were ·OH radicals (Figure 4d).


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Figure 4. pH-dependent MRI model switching of PEG/Fe5C2@Fe3O4 nanoparticles. (a, b) The longitudinal (a) and transverse (b) relaxation rates, respectively, of PEG/Fe5C2@Fe3O4 NPs dispersing at pH = 5.4 and pH = 7.4 environment. Inset: Corresponding MR images. (c) UV-Vis absorption spectra and photos of MB and H2O2 (50 μmol L-1) mixture after degradation by PEG/Fe5C2@Fe3O4 NPs at pH 5.4 and 7.4 for 24 h. Insets are a1: PEG/Fe5C2@Fe3O4 NPs + MB + H2O2 at pH 5.4; a2: MB + H2O2 at pH 5.4; b1: PEG/Fe5C2@Fe3O4 NPs + MB + H2O2 at pH 7.4; b2: MB + H2O2 at pH 7.4. (d) ESR spectra of different reaction condition with DMPO as the spin trap.

In Vitro Experiments with PEG/Fe5C2@Fe3O4 NPs


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We have shown above the potential of PEG/Fe5C2@Fe3O4 NP-activated ·OH generation based on the ferrous ion release in an acidic environment in the presence of H2O2. Being engulfed by acidic organelles, i.e., lysosomes/endosomes, is the trigger to produce ·OH in vitro. To substantiate the entrapment of NPs into endo/lysosomal compartments, PEG/Fe5C2@Fe3O4 NPs were conjugated with FITC and incubated with 4T1 cells for 4 h. By staining lysosomes with Lyso-Tracker Red, it showed that the NPs exhibited good colocalization with lysosomes (Figure 5a). Cellular TEM also confirmed that the PEG/Fe5C2@Fe3O4 NPs mainly accumulated in endocytic vesicles (Figure 5b). Compared with PEG/Fe5C2@C NPs, which maintained their morphologies after uptake, PEG/Fe5C2@Fe3O4 NPs broke into smaller particles within lysosomes, and the amounts of PEG/Fe5C2@Fe3O4 NPs were much lower, indicating the dissolution of PEG/Fe5C2@Fe3O4 NPs within cells. As the decomposition of Fe5C2@Fe3O4 NPs could lead to the release of Fenton reaction reactor, ferrous ions, the ROS levels of cells treated with PEG/Fe5C2@Fe3O4 NPs were then studied by 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). Because H2O2 is overproduced in the tumor extracellular microenvironment, a safe dose of H2O2 (50 μmol L-1) that has a negligible influence on cell viability was employed to simulate the real tumor environment (Figure S13). The fluorescence intensity was increased by prolonging the incubation time, and the PEG/Fe5C2@Fe3O4 NPs with H2O2 group and PEG/Fe5C2@Fe3O4 NPs treated group exhibited 2.5-fold and 60% higher ROS concentrations, respectively, after 9 h of culture compared to the control group (Figure 5c). The H2O2-added group also showed much more efficient ROS generation. The increased level of ROS in cells incubated with PEG/Fe5C2@Fe3O4 NPs and H2O2 was further visualized through the brighter green fluorescence of the cells on imaging (Figure 5d).


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Considering that the excessive production of ROS within cells could induce cell apoptosis, we further conducted cytotoxicity studies in different cell lines (Figure 5e and Figure 5f). The presence of 50 μmol L-1 H2O2 promoted cell proliferation (Figure S13), which is in accordance with the previous report,19 whereas the addition of PEG/Fe5C2@Fe3O4 NPs and H2O2 together, produced a significant NP dose-dependent cytotoxicity after 24 h of incubation. In contrast, little cytotoxicity could be observed under the same conditions when treating cells with PEG/Fe5C2@C NPs or PEG/Fe3O4 NPs (Figure S14, Figure S15 and Figure S16), indicating a better tumor cell inhibition effect based on PEG/Fe5C2@Fe3O4 NPs. The cell viability decreased slightly in the absence of H2O2, particularly at a low NP dosage, and little cytotoxicity was shown. Benefiting from the magnetic property of PEG/Fe5C2@Fe3O4 NPs, magnetic targeting was applied for the enhanced accumulation and cellular uptake of NPs. More blue spots by Prussian blue staining appeared after cells were exposed to a safe magnetic field (Figure S17). The magnetic field-assisted cellular uptake further promoted efficient ROS generation (Figure S18). Consequently, the survival rate of cells treated with PEG/Fe5C2@Fe3O4 NPs in the presence of H2O2 and a magnetic field was significantly decreased (Figure 5e). Interestingly, the cytotoxicity of PEG/Fe5C2@Fe3O4 NPs in the presence of H2O2 to 4T1 cancer cells was greater compared to its cytotoxicity to noncancerous 293T cells, probably because cancer cells are more vulnerable to ROS (Figure S19).12 In addition, PEG/Fe5C2@Fe3O4 NPs alone presented relatively low cytotoxicity in normal cell lines such as 293T, L929, and L02 cells (Figure 5f), indicating their biosafety for potential in vivo applications. Compared with cancerous 4T1 cells, normal cells showed less cytotoxicity from PEG/Fe5C2@Fe3O4 NPs alone (Figure S20). This can be ascribed to the intracellular overexpression of H2O2 and sensitivity to ROS in cancerous cells.42 All the above results confirmed that Fe5C2@Fe3O4 NPs could act as ROS generators that


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selectively induce cancer cell apoptosis in a H2O2-overproducing environment under magnetic guidance.

Figure 5. In vitro experiments with PEG/Fe5C2@Fe3O4 NPs. (a) Fluorescence image of 4T1 cells treated with PEG/Fe5C2@Fe3O4-FITC NPs for 4 h. Nuclei of live cells were stained with Hoechst 33342 and lysosomes were stained with Lyso-Tracker Red. Scale bars are 15 μm. (b) TEM image of 4T1 cells treated with (b1, b2) PEG/Fe5C2@Fe3O4 NPs and (b3, b4) PEG/Fe5C2@C NPs for 4 h. (b2) and (b4) are the high magnification for the rectangle area in (b1)


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and (b3). (c) Time-dependent fluorescent intensity from DCFH-DA labeled 4T1 cells by PEG/Fe5C2@Fe3O4 NPs with or without H2O2. (n = 3, mean ± s.d., **p < 0.01, and ***p < 0.001). (d) Fluorescence images of DCFH-DA labeled 4T1 cells treated by PEG/Fe5C2@Fe3O4 NPs under different incubation condition. Scale bars are 75 μm. (e) Cell viability studies on 4T1 cells model after incubation with PEG/Fe5C2@Fe3O4 NPs or PEG/Fe5C2@Fe3O4 NPs + H2O2 (50 μmol L-1) with or without magnetic field. (f) Cell viability studies on 293T cells, L929 cells and L02 cells model after incubation with PEG/Fe5C2@Fe3O4 NPs. (n = 6, mean ± s.d., *p < 0.05, **p < 0.01, and ***p < 0.001).

In Vivo Imaging and Experiments with PEG/Fe5C2@Fe3O4 NPs Encouraged by the fascinating magnetic properties and in vitro magnetic targeting based on PEG/Fe5C2@Fe3O4 NPs, we then conducted a magnetic-guided tumor enrichment experiment in 4T1 tumor-bearing mice by MRI. Mice were injected with PEG/Fe5C2@Fe3O4 NPs intravenously (i.v.) at a dose of 10 mg Fe kg−1 (metal to body weight), and MR images were taken before and one-day postinjection (Figure 6a and Figure S21). A significant decrease of 30.5% in the T2 MRI signal intensity was observed after injection of PEG/Fe5C2@Fe3O4 NPs by fixing a magnet on the tumor, demonstrating efficient tumor magnetic targeting and retention. To further investigate whether NPs accumulated at the tumor can be degraded to ferrous ions, MRI signal change was monitored after intratumoral (i.t.) injection of PEG/Fe5C2@Fe3O4 NPs. The T2-weighted MRI signal decreased to 70.3% immediately postinjection and recovered to 83.2% 1 day later, indicating that NPs were partly degraded or metabolized (Figure S22). Moreover, an enhancement in the T1-weighted MRI signal from 99.1% to 123.7% was observed within 1 day


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(Figure 6b), confirming the dissolution of NPs and release of Fe2+. In contrast, by i.t. injection of PEG/Fe3O4 NPs, only the decrease in T2-weighted MRI signal was observed, without an obvious T2 signal recover and T1 signal changes 1 day postinjection (Figure S23), primarily due to their low pH sensitivity. Although some reports have indicated that Fe2+ could be easily oxidized to Fe3+,47 it is fortunate that the hypoxic environment and overproduction of reductive glutathione at the tumor site can decrease the oxidation of ferrous ions, providing a highly catalytic environment for the Fenton reaction for ROS generation. We next investigated the in vivo tumor ROS concentration due to overproduced H2O2. Brighter green fluorescence was emitted in the group that had been subjected to i.t. injection of PEG/Fe5C2@Fe3O4 NPs after staining with DCFH-DA, with the fluorescence intensity increased by 12.4-fold compared with that of the control group (Figure 6c and Figure S24). This intensity level was increased 5.4 times for the i.v. injection group with the assistance of magnetic targeting, which resulted from the accumulation of more NPs in the tumor, while the intensity was only 2.1 times higher for the group treated with only i.v. injection of NPs. The high ROS intensity led to tumor cell apoptosis and even necrosis. As shown in Figure 6d, obviously degenerative changes of coagulative necrosis with condensed cell nuclei were observed in both the i.t. injected group and the i.v.-injected with magnetic targeting group, whereas these features were not found in the control groups. We next assessed the efficacy of inhibiting tumor growth. The mice were injected once daily every three days, each with 50 mg Fe kg-1, and tumor volume was monitored one-day postinjection. As expected, mice treated with i.t. injection and magnetic-assisted i.v. injection showed a considerable delay in tumor growth. Notably, in the i.t. injection mice, parts of the tumors were completely inhibited within 19 days, as a result of efficient iron ionization and ·OH generation for the intratumoral Fenton reaction


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(Figure 6e and Figure S25). In contrast, mice i.v. injected with NPs exhibited much lower therapeutic effects due to the low iron concentration. No mice died during the course of therapy, and no abnormality was observed in the time-dependent body weight curves (Figure 6f). Mice were then sacrificed on the 19th day for examination of any effects on major organs by H&E staining (Figure S26), and the results revealed no significant abnormality in any major organ. Compared with PEG/Fe3O4 NPs, the therapeutic effect from i.t. injection of PEG/Fe5C2@Fe3O4 NPs was a bit higher, primarily due to their higher pH sensitivity (Figure S27 and Figure S28). The slight inferiority of PEG/Fe5C2@Fe3O4 NPs than Fe2+ ions in tumor inhibition can be attributed to the inadequacy of iron release from PEG/Fe5C2@Fe3O4 NPs at tumor, which is in accordance with the results from Figure 3b and Figure S10. Nevertheless, neither T1 nor T2 MRI signal changes were observed post i.t. injection of Fe2+ ions within one day, since there is no chemical change in tumor (Figure S29), which resulted in the infeasibility for the visible therapy by Fe2+ ions. Therefore, the combination of effective tumor inhibition and switchable T2/T1 signal conversion in MRI endow PEG/Fe5C2@Fe3O4 NPs great promise in imaging detectable therapeutic agents. Biodistribution is a crucial factor in determining the safety of biomedical agents. To investigate this parameter, IR783-labeled PEG/Fe5C2@Fe3O4 (PEG/Fe5C2@Fe3O4-IR783) NPs were intravenously injected into mice followed by in vivo NIR optical imaging of tumors and major organs. As shown in Figure S30A, PEG/Fe5C2@Fe3O4-IR783 NPs gradually accumulated at the tumor site, and a preferential accumulation in the tumor was realized by locating a magnet on the tumor, with a much stronger fluorescence intensity observed. The ex vivo fluorescence intensity was also investigated 24 h postinjection, which suggested that NPs were mainly accumulated in the tumor, liver and kidney (Figure S30B). High fluorescence signal from liver


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was attributed to the capture of NPs by reticuloendothelial system (RES), and the accumulation in kidney may be due to the excretion of small-sized molecules.48,49 A slight promotion of tumor enrichment and reduction of the accumulation of NPs in bystander organs was observed with the assistance of magnetic targeting, which shows promise for improving the biosafety of PEG/Fe5C2@Fe3O4 NPs. To quantitatively determine the biodistribution of PEG/Fe5C2@Fe3O4 NPs, iron concentrations in tumor and major organs were measured 24 h after i.v. administration (10 mg Fe kg-1). The result showed that NPs were mainly distributed in liver and spleen, and magnetic targeting was able to improve their tumor accumulation (Figure S31), which was accordance with the fluorescence result. Although further investigations are still needed to confirm PEG/Fe5C2@Fe3O4 as a safe and biocompatible material, our preliminary results suggest that it may be an ideal candidate for switchable MRI-guided cancer therapy.


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Figure 6. In vivo imaging and experiments with PEG/Fe5C2@Fe3O4 NPs. (a) Representative T2weighted MR images of 4T1 tumor-bearing mice before and one day after i.v. injection of PEG/Fe5C2@Fe3O4 NPs with or without magnetic targeting. The tumor sites are circled by a yellow dashed line. Figure at the top right corner is the pseudo-color image of the tumor site. Scale bars are 5 mm. (b) Representative T1-weighted MR images of 4T1 tumor-bearing mice (b1) before, (b2) immediately post, and (b3) one day after i.t. injection of PEG/Fe5C2@Fe3O4 NPs. The tumor sites are circled by a yellow dashed line. Figure at the top right corner is the pseudocolor image of the tumor site. (b4) Intensity changes of T1-weighted MR signal immediately or one day after i.t. injection. Scale bars are 5 mm. (n = 3, mean ± s.d., **p < 0.01). (c)


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Fluorescence image of DCFH-DA labeled tumor by the frozen section in different groups. Scale bars are 25 μm. (d) H&E staining of 4T1 tumor sections in different groups. Scale bars are 500 μm. (e, f) Time course change in (e) the relative tumor volume and (f) the body weight after different treatments. (n = 5, mean ± s.d., ***p < 0.001).

Conclusions In summary, we have developed a switchable MRI-guided cancer therapeutic agent based on ROS generation by Fe5C2@Fe3O4 NPs. These NPs are pH-sensitive, releasing ferrous ions in acidic tumor environments, and the discharged Fe2+ ions disproportionate the H2O2 that is overproduced at tumor sites to generate ·OH radicals for effective cancer therapy. Moreover, they have high magnetic properties, which are beneficial as they allow visualization of tumor aggregation through magnetic targeting and T2-weighted MRI. The effective tumor orientation and ROS generation were confirmed through both in vitro and in vivo experiments, which showed excellent therapeutic efficacy with low toxicity. In addition, the dissolution of Fe5C2@Fe3O4 NPs in the low-pH region reduces the T2 signal on MRI, and the release of ferrous ions raises the T1 signal, providing an MRI-supervised cancer therapy. These Fe5C2@Fe3O4 NPs are the pioneering paradigm of the application of iron carbide for tumor regression based on the selective catalysis of the Fenton reaction without the need for external energy input, providing a visible strategy for efficient and specific cancer therapy. However, Fe5C2@Fe3O4 NPs are a rising type of magnetic material, and their biomedical applications are just emerging. Challenges still remain for further clinical applications despite their high performance. For instance, the therapeutic mechanisms of magnetic NPs are


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complicated. Taking FDA-approved iron oxide NPs for example, both their enzyme-like activity and induction of pro-inflammatory macrophage polarization cause tumor inhibition.50,51 Is the high curative effect of Fe5C2@Fe3O4 NPs also due in part to these factors as well as ROS generation? This requires further deep study. In addition, it seems that Fe5C2@Fe3O4 NPs at a high concentration have effects on cell proliferation in vitro, while from the in vivo results of body weight and H&E staining of major organs, no noticeable side effects were observed from our injected dose. Therefore, the biodegradation, metabolization, pharmacokinetics and longterm biocompatibility of Fe5C2@Fe3O4 NPs also need to be investigated in detail in the future. Furthermore, we still aim to improve Fe5C2@Fe3O4 NPs tumor-targeting properties by further surface modifications as well as optimize hydroxyl radical concentrations to reduce the side effects to normal cells by precisely controlling NPs’ activity and concentration. Nevertheless, the good performance in ROS-driven cancer therapy and switchable MRI-based monitoring of the curing process described in this manuscript shows that Fe5C2@Fe3O4 NPs are great candidate as imaging-guided cancer therapeutic agents.

Materials and Methods Materials Iron carbonyl (Fe(CO)5, 99%), oleylamine (OAm), oleic acid (OAc, 90%), 1-octadecene (ODE, 90%), cetyltrimethylammonium bromide (CTAB, 99%) and ammonium bromide (NH4Br, 99%) were purchased from Alfa Aesar. Hexane(C6H14), ethanol (C2H6OH) and hydrogen peroxide (H2O2) of analytic grade were from the Juhua Group Factory, China. 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG-NH2) was


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purchased from toyongbio corporation, china. All chemicals were used as received without further purification. Synthesis and Modification of Fe5C2@Fe3O4 NPs and Fe5C2@C NPs Fe5C2@Fe3O4 NPs were synthesized following our previously reported method.33 First, bcc-Fe NPs were synthesized by the following steps. 62.5 mmol octadecene (ODE), 0.1 mmol NH4Br and 1 mmol oleylamine (OAm) were mixed, magnetically and degassed at 100 ℃ for 2 h before being heated to 180 °C. At the temperature of 180 °C, 5 mmol Fe(CO)5 was injected under the protection of Ar. The reaction mixture was then kept for 30 min until the color turn to black. When cooling the resultant solution to 140 °C, 1 mmol oleic acid (OA) and hexane (0.2 mL) were added via a syringe, and then aged for another 30 min before cooling down to room temperature. The harvest bcc-Fe NPs were washed and collected with ethanol and hexane. For further carbonizing bcc-Fe NPs to Fe5C2@Fe3O4 NPs, octadecylamine (ODA, 37.5 mmol) with NH4Br (0.05 mmol) was degassed at for at 120 °C 1 h. Then, the resulted bcc-Fe NPs (5 mmol, in 10 mL hexane) were added via a syringe and the reaction solution was heated at 130 °C for another 30 min before it was heated to 300 °C for 30 min. The solution was then cooled to room temperature and washed with ethanol and hexane three times. Fe5C2@C NPs were synthesized following our previously reported method.42 A mixture of ODA (14.5 g) and CTAB (0.113 g) was degassed under a flow of N2, and heated to 120 °C, followed by injecting Fe(CO)5 (3.6 mmol) under a N2 blanket. The mixture was heated to 180 °C at 10 °C min-1 and kept for 10 min, subsequently further heated to 350 °C at 10 °C min-1 and kept for 10 min before it was cooled to room temperature. The product was washed with ethanol and hexane and collected for further characterization. To convert NPs into water solution with better biocompatibility (to form PEG/Fe5C2@Fe3O4), NPs were derived using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-


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N-[amino(polyethylene glycol)-2000] (DSPE-PEG-NH2). 20 mg DSPE-PEG-NH2 was dissolved in 15 mL chloroform and added dropwise to chloroform solution containing 5 mg Fe5C2 NPs. The solution was stirred overnight under Ar protection, followed by evaporating chloroform by rotary evaporation. Then add water and dialyze for 24 h to remove excess DSPE-PEG-NH2. Characterization Transmission electron microscopy was carried out on a FEI Tecnai T20 microscope. Highresolution TEM (HRTEM) was carried out on a FEI Tecnai F20 microscope. X-ray diffraction patterns were recorded on a Rigaku DMAX-2400 X-ray diffractometer equipped with Cu Kα (λ = 1.5405 Å) radiation. Magnetization was measured by a superconducting quantum interference device (SQUID). X-ray photoelectron spectroscopy (XPS) measurements were carried out on an Axis Ultra imaging photoelectron spectrometer (Kratos Analytical Ltd.). Dynamic light scattering was measured using a particle size analyzer (ZetaPALS, Brookhaven Instruments, Holtsville, NY). The concentrations of Fe were quantified using an inductively coupled plasmaatomic emission spectrometer (ICP-AES, Profile, Leeman, USA). pH-Dependent Ferrous Ion Release 1 mL of PEG/Fe5C2@Fe3O4 NPs or PEG/Fe5C2@C or PEG/Fe3O4 NPs solution with Fe concentration of 2 mg mL-1 was sealed in a dialysis bag (MCWO: 1000 Da), and immersed in 20 mL buffer media (PBS) at different pH values (7.4, 6.5 and 5.4) in a centrifuge tube. PBS was composed of 0.2 mol L-1 sodium dihydrogen phosphate and 0.2 mol L-1 sodium hydrogen phosphate, and the pH value of buffer was adjusted by the ratio of sodium dihydrogen phosphate to sodium hydrogen phosphate. Centrifuge tubes were then shaken with a speed of 200 rpm at 37 °C. At given intervals, 1 mL of buffer was collected and analyzed the Fe concentration by ICP-


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AES. 1 mL fresh buffer medium was returned. Fe released (R(Fe,n)) was calculated by the following equation: 𝑛―1

𝑅(𝐹𝑒,𝑛)(%) =

20𝐶𝑛 + ∑𝑖 = 1 𝐶𝑖 2∗1

∗ 100%

Where Cn is the iron concentration tested in nth collection, Ci is the iron concentration in the ith collection. Unit of Cn and Ci are mg mL-1. To confirm the iron ion released was ferrous ion, potassium ferricyanide and potassium thiocyanate were applied. Briefly, PEG/Fe5C2@Fe3O4 NPs were dispersed in a buffer solution with pH value of 5.4 and 7.4, and the NPs concentration was into 12-well plates. Then, potassium ferricyanide and potassium thiocyanate were added respectively, and the solutions incubated for 24 h. After centrifugation to remove undissolved NPs, photos of the products were taken. In Vitro MRI Test The in vitro MRI tests were conducted on a 3.0 T clinical MRI instrument (Philips). To test the in vitro longitudinal relaxation rate r2, PEG/Fe5C2@Fe3O4 NPs at the iron concentration from 0.003 mmol L-1 to 0.021 mmol L-1 with the interval of 0.003 mmol L-1 were dispersed in 1 mL of solution with pH 5.4 and 7.4 containing 1 wt% agarose gel respectively and placed in centrifuge tubes (1.5 mL). MR images were acquired using a T2-weighted sequence with the following parameters: Repetition time (TR) = 500 ms; echo time (TE) from 12.25 ms to 196 ms, with the interval of 12.25 ms; field of view (FOV) = 180 mm; slice thickness = 2 mm; NEX = 1. To test the in vitro T1 relaxation rate r1, NPs at the iron concentration from 0.044 mmol L-1 to 0.84 mmol


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L-1 with the interval of 0.114 mmol L-1 were dispersed in 1 mL of solution with pH 5.4 and 7.4 containing 1 wt% agarose gel respectively and placed in centrifuge tubes (1.5 mL). The parameters of a T1-weighted sequence were as follows: TE = 41.54 ms; TR = 2300 ms and 920 ms; FOV = 170 mm; slice thickness = 3 mm; NEX = 1. T1/T2-weighted images were acquired by using the post-processing software in Philips DICOM Viewer, and the selected region of interests (ROIs) in the T1/T2-weighted images were measured with the same size to obtain the signal intensities for each condition. Detection of ROS PEG/Fe5C2@Fe3O4 NPs (1.5 mg) were added in 5 mL of methylene blue (MB) solution (5 mg L-1) with 50 μmol L-1 of H2O2. After 24 h incubation, the MB aqueous solution was centrifuged to remove unreacted NPs. UV-Vis spectroscopy was measured on the supernatant. ESR spectroscopy was used to confirm the ROS generated of was ·OH. 5,5-Dimethyl-1pyrroline N-oxide (DMPO) was applied as a spin trap for the hydroxyl radical. PEG/Fe5C2@Fe3O4 NPs with or without 50 μmol L-1 of H2O2 at pH 5.4 were mixed with 40 μL of DMPO solution (100 mmol L-1). 2 μL of the mixture was immediately injected into a quartz capillary, and X band ESR spectra were then measured (Bruker A300). The parameters were set as follows: microwave frequency = 9.423 GHz, microwave power = 10.12 mW, modulation frequency = 100.00 kHz and modulation amplitude = 1.00 G. Cell Culture All cell lines were obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Science. All cell-culture-related reagents were purchased from Invitrogen. Cells were


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cultured in DMEM culture medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under 5% CO2 with 100% humidity. Confocal Laser Scanning Microscopy Study The as-prepared of PEG/Fe5C2@Fe3O4 NPs was labeled with fluorescein isothiocyanate (FITC) (λex = 488 nm, λem = 517 nm) defined as PEG/Fe5C2@Fe3O4-FITC. 4T1 cells (2 × 104 cells per well) were seeded into a 24-well plate. When the cell confluence reached 80%, 100 μg mL-1 of PEG/Fe5C2@Fe3O4-FITC NPs were added into the culture medium and co-cultured with cells at 37 °C for 4 h. After that, 4T1 cells were counterstained with Lyso-Tracker Red (λex = 577 nm, λem = 590 nm) for 30 min and Hoechst 33342 (λex = 350 nm, λem = 461 nm) for 15 min. Finally, the cells were washed three times with PBS to remove the NPs that have not been endocytic. The images were taken by using a confocal microscope (Leica SP8). Cellular TEM Image 4T1 cells were incubated with PEG/Fe5C2@Fe3O4 NPs or PEG/Fe5C2@C NPs ([Fe] = 10 μg mL-1) for 4 h, washed with DPBS three times, and then collected by centrifugation. The cell pellets were fixed in DPBS solution containing 2% glutaraldehyde and 2.5% paraformaldehyde for 2 h, post fixed in 1% osmium tetroxide, washed by sodium cacodylate buffer, dehydrated with gradient alcohol, replaced by propylene oxide, and embedded in Epon 812. Semithin sections (1 μm) were cut, stained by methylene blue, and localized by a microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a JEM-1400 electron microscope. Intracellular ROS Formation


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4T1 cells were placed at a density of 2 × 104 cells per well into 24-well plates and were incubated for 12 h. Cells were loaded with 2’,7’ Dichlorodihydrofluorescein diacetate (DCFHDA, 10 µmol L-1) for 30 min and then washed with PBS for three times. Then, cells were treated by PEG/Fe5C2@Fe3O4 NPs ([Fe] =100 μg mL-1) or PEG/Fe5C2@Fe3O4 NPs with 50 µmol L-1 H2O2. The intensity of fluorescence was recorded every 15 min over a period of 9 h via excitation of 488 nm and emission at 538 nm by BioTek ELx800. For the fluorescent microscopic examination, 4T1 cells with the cell density of 5 × 104 cells per were plated onto coverslips in 12-well plates overnight. Then, cells were pre-stained with 10 µmol L-1 DCFH-DA for 30 min and washed with PBS to remove the free DCFH-DA. Later, the cells were incubated with PEG/Fe5C2@Fe3O4 NPs ([Fe] = 100 μg mL-1) or PEG/Fe5C2@Fe3O4 NPs with 50 µmol L-1 H2O2 or PEG/Fe5C2@Fe3O4 NPs with 50 µmol L-1 H2O2 and a magnet for 4 h before they were washed with PBS for three times. The images were acquired by a Leica SP8 confocal microscope with excitation at 488 nm and emission from 525 nm. Intracellular Magnetic Targeting 4T1 cells were plated in 24-well plates (2 × 104 cells per well) for 12 h, followed by adding of PEG/Fe5C2@Fe3O4 NPs ([Fe] = 10 μg mL-1) for another incubation of 4 h. In the magnetic target group, a magnet was placed at the bottom of the culture plate. Then, cells were fixed with 4% paraformaldehyde and stained with Prussian blue dye (2% of potassium ferrocyanide and 2% of HCl, V/V = 1:1) for 30 min and 0.01% neutral red for another 30 min. The images were observed under optical microscope (Nikon DS-Ri2). Cytotoxicity Assays


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The in vitro cytotoxicity was evaluated by standard MTS assay. 4T1, 293T, L02 and L929 cells were seeded into 96-well cell culture plates at 5 × 103 cells per well and incubated overnight at 37 ℃ under 5% CO2. To evaluate the influence of H2O2, after removing the culture medium, fresh culture medium with H2O2 concentration of 12.5, 25, 50, 100, 200 and 400 μmol L-1 were added followed by further incubation of 24 h. To evaluate the influence of PEG/Fe5C2@Fe3O4 NPs, fresh culture medium containing PEG/Fe5C2@Fe3O4 NPs at [Fe] of 50, 100, 150, 200, 250, 300, 350, 400 μg mL-1 were added for incubation of 24 h. To simulate the tumor microenvironment, culture medium containing 50 μmol L-1 H2O2 and various concentrations of PEG/Fe5C2@Fe3O4 NPs were added. To simulate the magnetic targeting group, a magnet was put under the culture plate. After incubation for 24 h, the culture media were removed and cells were washed three times with PBS. MTS solution (2 mg mL-1) was then added into each well for another 4 h incubation. The absorbance of suspension was measured by an ELISA reader (Tecan m200) at 490 nm. Animal Modal All experiments involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University, Beijing China. 6-week female Balb/c mice, with an average weight of 17 g, were provided by the Beijing Center for Disease Control and Prevention, Beijing, China. Mice were injected with 5 × 105 4T1 cells (0.2 mL cells in DMEM culture medium without FBS) subcutaneously at the right axillary region. Living Animal Tumor Study by MRI


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When the tumor volumes reached about 200 mm3, six selected mice were randomly divided into two groups with each group size of three, and each mouse was administered an intravenous injection (i.v.) of PEG/Fe5C2@Fe3O4 NPs (10 mg Fe kg-1) via tail vein. A permanent magnet was located at the tumor site in the magnetic targeting group mouse (n = 3). MR images were acquired before and 1 day after injection with an animal-specific body coil. Data acquisition was performed after the mice were anesthetized by isoflurane (1.0 ~ 2.0%) in oxygen. The T2-map images were obtained in a 7 T MRI scanner (Bruker), and the sequence is TR = 3000 ms, TE = 50 ms, slice thickness = 1.0 mm. To evaluate the iron release and MRI mode switching from PEG/Fe3O4 NPs, PEG/Fe5C2@Fe3O4 NPs and Fe2+ ions, nine selected mice were randomly divided into three groups when the tumor volume was 100 mm3, and each group were of three mice. PEG/Fe3O4 NPs, PEG/Fe5C2@Fe3O4 NPs or Fe2+ ions (6 mg Fe kg-1) were intratumoral (i.t.) injected. MR images were acquired before, immediately post, and 1 day after injection by a 3 T clinical MRI scanner (Philips). T1-map sequence is TR = 7.8 ms, TE = 3.3 ms, slice thickness = 1.0 mm; and the T2-map sequence is TR = 2600 ms, TE = 362.5 ms, slice thickness = 0.8 mm. T1/T2-weighted images were acquired by using the post-processing software in Philips DICOM Viewer, and the selected region of interests (ROIs) in the T1/T2-weighted images were measured with the same size to obtain the signal intensities for each condition. Tumor Inhibition and In Vivo Toxicity Assay When the tumor volume reached to about 100 mm3, 28 mice were randomly divided into 4 groups: (1) mice were intratumorally injected of PEG/Fe5C2@Fe3O4 NPs; (2) mice were intravenously injected of PEG/Fe5C2@Fe3O4 NPs with a magnet located at the tumor site; (3)


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mice were intravenously injected of PEG/Fe5C2@Fe3O4 NPs; (4) control group. All groups were injected once daily every three days in the period of 18 days with the dosage of 50 mg Fe kg-1 and tumor volume was monitored 1-day post injection. Tumor volumes and body weight were monitored 1-day post injection during the treatment (n = 5). Tumor volume was calculated according to the formula of (a × b2)/ 2, where a and b are the long and short diameters of the tumor, respectively. Three days after injection, one mouse from each group was euthanized, and tumor was harvested from the necropsy; At given intervals of 19 days, mice from each group were euthanized and major visceral organs (heart, liver, spleen, lung, and kidney) were recovered, followed by fixing with 10% neutral buffered formalin. After the organs were embedded in paraffin and sectioned at 5 μm, hematoxylin and eosin (H&E) staining was performed. The slides were observed under optical microscope (Nikon ECLIPSE Ni-U). Quantitative Analysis of the ROS Generation in Tumors At the 3rd day post injection, one mouse from each group was euthanized, and tumors were harvested from the necropsy. The tumor tissues were firstly embedded and frozen by an OCT (Jung, Tissue freezing medium, Leica). Cross sections of 10 μm thickness were cut using a cryomicrtome (Leica), staining with DCFH-DA, 10 µmol L-1 for 30 min and mounted on the glass slides. The slides were then observed under a confocal microscope (Leica SP8, λex = 488 nm; λem = 525 nm). Biodistribution Studies Noninvasive NIR imaging was used to visually monitor biodistribution and accumulation in


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tumor of PEG/Fe5C2@Fe3O4 NPs. PEG/Fe5C2@Fe3O4 NPs were labeled with IR783, a kind of NIR dye, through electrostatic interaction. Briefly, 5 mg PEG/Fe5C2@Fe3O4 NPs and 5 mg IR783 were dispersed into 10 mL PBS, followed by stirring overnight. Fe5C2@Fe3O4-IR783 NPs were collected by centrifugation (12000 rpm, 5 min). The model mice were divided into two groups, one group of mice was intravenously injected with IR783-loaded Fe5C2@Fe3O4 NPs, the other was intravenously injected with IR783-loaded PEG/Fe5C2@Fe3O4 NPs plus magnet, with the same dosage of IR783 (2 mg kg-1). After 1, 3, 6, and 24 h, Xenogen in vivo imaging system IVIS Spectrum (Xenogen, USA) was used to record the results, with an excitation bandpass filter at 700 nm and an emission at 830 nm. After injection for 24 h, heart, liver, spleen, kidney, and tumor tissues of each group were taken out and recorded fluorescence intensity. In vivo fluorescence images were acquired by using the post-processing software in Living Image. To qualitatively investigate the biodistribution of PEG/Fe5C2@Fe3O4 NPs, 6 mice were assigned into two groups randomly. When the tumor volume reached to about 100 mm3, mice were intravenously injected of PEG/Fe5C2@Fe3O4 NPs (10 mg Fe kg-1). In the magnetic targeting group, a magnet was located at the tumor site. Mice were sacrificed after heart perfusion at time point of 24 h with major organs (heart, liver, spleen, lung and kidneys) and tumors dissected, rinsed and weighted. The in vivo biodistribution of Fe element was then measured by ICP-AES and calculated as Fe percentage over administrated dose per gram of tissues. Statistical Analysis


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Quantitative data were expressed as mean ± s.d. Statistical comparisons were conducted by using student’s two-tailed t test. Values with p < 0.05 were considered statistically significant (∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.001). Data Availability All other remaining data are available within the article and supplementary files, or available from the authors upon request.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . TEM, XPS analysis, DLS results, accumulative iron ion release curve, room-temperature magnetic hysteresis loops, cell viability of 4T1 cells incubated with H2O2, cell viability of 4T1 cells incubated with PEG/Fe5C2@C NPs and PEG/Fe3O4 NPs, Prussian blue staining, cell viability studies on 293T, L929, L02 and 4T1 cells incubated with PEG/Fe5C2@Fe3O4 NPs, intensity change of T2-weighted MR signal, H&E stained images, in vivo NIR imaging, and the biodistribution of Fe in main tissues and tumors (PDF).

Author Information


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Corresponding Authors * E-mail: [email protected]. (X. Mou) * E-mail: [email protected]. (S. Che) * E-mail: [email protected]. (Y. Hou) Author Contributions ¶

These authors contributed equally to this manuscript.

J. Yu, F. Zhao and W. Gao designed the project. J. Yu, F. Zhao, W. Gao, X. Yang and Y. Ju performed the experiments and analyzed the results. X. Mou, S. Che and Y. Hou provided useful suggestions to this work. J. Yu and F. Zhao wrote the manuscript.

Acknowledgment This work was supported in part by the National Natural Science Foundation of China (51602285, 81701821, 51672010, 81421004), Natural Science Foundation of Beijing Municipality

(L172008),

Young

Elite

Scientist

Sponsorship

Program

by

CAST

(2017QNRC001), Fund of Key Laboratory of Advanced Materials of Ministry of Education (53220330118), and the Fundamental Research Funds for the Provincial Universities of Zhejiang (RF-A2019004).

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