Biomaterials 38 (2015) 1e9

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FeS nanoplates as a multifunctional nano-theranostic for magnetic resonance imaging guided photothermal therapy Kai Yang a, b, *, Guangbao Yang b, Lei Chen a, Liang Cheng b, Lu Wang b, Cuicui Ge a, Zhuang Liu b, ** a School of Radiation Medicine and Protection & School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Suzhou Nano Science and Technology & Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, Suzhou, Jiangsu 215123, China b Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2014 Accepted 18 October 2014 Available online

In this work, we develop magnetic iron sulfide (FeS) nanoplates as a theranostic agent for magnetic resonance (MR) imaging-guided photothermal therapy of cancer. FeS nanoplates are synthesized via a simple one-step method and then functionalized with polyethylene glycol (PEG). The obtained PEGylated FeS (FeS-PEG) nanoplates exhibit high NIR absorbance together with strong superparamagnetism. The r2 relaxivity of FeS-PEG nanoplates is determined to be 209.8 mM-1S-1, which appears to be much higher than that of iron oxide nanoparticles and several types of clinical approved T2-contrast agents. After intravenous (i.v.) injection, those nanoplates show high accumulation in the tumor as revealed by MR imaging. Highly effective photothermal ablation of tumors is then achieved in a mouse tumor model upon i.v. injection of FeS-PEG at a moderate dose (20 mg/kg) followed by 808-nm NIR laser irradiation. Importantly, it has been found that PEGylated FeS nanoplates after systemic administration could be gradually excreted from major organs of mice, and show no appreciable toxicity to the treated animals even at a dose (100 mg/kg) 5 times as high as that used for imaging & treatment. Our results demonstrate that PEGylated FeS nanoplates may be a promising class of theranostic nano-agents with a good potential for future clinical translation. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Iron sulfide nanoplates Magnetic resonance imaging Photothermal therapy Cancer theranostics

1. Introduction Photothermal therapy (PTT), as a non-invasive cancer therapeutic method, has attracted great attention in recent years [1e3]. PTT mainly depends on photo-absorbing nanomaterials to generate heat under laser irradiation to burn cancer cells while do not affect normal cells and tissues [4e8]. A large verity of photothermal nano-agents, including gold nano-structures, nano-carbons, metal sulfide nanoparticles, as well as many organic nanoparticles, have been developed in re-cent years for photothermal ablation of cancers [1e3, 8e26]. On the other hand, to optimize the treatment planning and monitor the therapeutic responses, imaging guided

* Corresponding author. School of Radiation Medicine and Protection & School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Suzhou Nano Science and Technology & Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, Suzhou, Jiangsu 215123, China. ** Corresponding author. E-mail addresses: [email protected] (K. Yang), [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.biomaterials.2014.10.052 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

therapy has been widely applied in biomedical research and clinical trials [27e30]. Imaging guided PTT, in particular, could not only illustrate the tumor locations, sizes and shapes to ensure the effective exposure of tumors to the laser during treatment, but also determine the best time timing of laser irradiation when the photothermal agent reaches the peaked level in the tumor [1,28]. Among various imaging modalities, magnetic resonance (MR) imaging is one of the most widely applied and useful imaging techniques in the clinic [31e33]. On the one hand, MR imaging can provide high spatial and temporal resolution for whole body imaging [27,34e36]. On the other hand, MR imaging, different from Xray computed tomography (CT) and nuclear imaging, is a relatively safe imaging method without inducing any ionizing radiation to the patients [34,37]. In recent years, many groups including ours have developed various types of theranostic agents by incorporating magnetic nanoparticles with light-absorbing nanostructures to obtain multifunctional nanocomposites, for MR-imaging guided photothermal therapy [1,7,12,38]. For example, in a recent study by Tian et al., Fe3O4@Cu2xS core-shell nanoparticles are synthesized and used for MR imaging and photothermal ablation of tumors in

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animals [39]. In a latest work, Hou and co-workers demonstrated that Fe5C2@C nanoparticles with carbon shells could serve as a multifunctional MR imaging probe as well as a photothermal agent [40]. However, the synthesis procedures usually involve multiple steps and need rather careful controls for those composite nanoparticles, which, on the other hand, may have complicated degradation and excretion behaviors in biological systems as different components may behave differently in vivo. The development of theranostic agents for imaging-guided PTT with a single component would thus be of great interests. Although various types of metal sulfide nanostructures (e.g. CuS, MoS2, WS2, etc.) with strong NIR absorbance have been extensively explored as photothermal agents [20,23,29], the development of metal sulfide nanostructures with a single inorganic component for MR imaging guided PTT, has not yet been reported to our best knowledge. Considering the fact that several formulations of iron oxide nanoparticles (ferumoxsil, ferrixan and ferumoxide) with appropriate coatings have already been approved as safe T2-MR contrast agents for clinical diagnosis, in this work, we for the first time develop magnetic iron sulfide (FeS) nanoplates as a theranostic agent for MR imaging-guided photothermal therapy

of cancer (Fig. 1a). FeS nanoplates are synthesized via a simple one-step method and then functionalized with polyethylene glycol (PEG). The obtained PEGylated FeS (FeS-PEG) nanoplates exhibit high NIR absorbance together with strong magnetic property. The r2 relaxivity of FeS-PEG nanoplates is determined to be 209.8 mM1 S1, which appears to be much higher than that of clinical approved T2-contrast agents (72 mM1 S1 for ferumoxsil, 151 mM1 S1 for ferrixan, and 98.3 mM1 S1 for ferumoxide). After intravenous (i.v.) injection, those nanoplates show high accumulation in the tumor as revealed by MR imaging. Highly effective photothermal ablation of tumors is then achieved in a mouse tumor model upon i.v. injection of FeS-PEG at a moderate dose (20 mg/kg) followed by 808-nm NIR laser irradiation. More importantly, it is uncovered that i.v. injected FeS-PEG at an extremely high dose, 100 mg/kg, which is five times of the treatment dose, affords no appreciable toxicity to mice as evidenced by blood analysis and careful histology examination. The Fe concentrations in the major organs of mice decreased to be close to the normal levels in those mice, suggesting the gradual clearance of FeS after administration. Our results present the great promise of FeS nanoplates as a safe, multi-functional theranostic agent for

Fig. 1. Preparation and characterization of FeS nanoplates. (a) A scheme showing the preparation of PEGylated FeS nanoplates. As-made FeS nanoplates with hydrophobic TOPO coating were functionalized by different layers of polymer coatings and then conjugated with PEG. (b) TEM images of as-made FeS nanoplates. Inset is a high-resolution TEM image of a FeS nanoplate. (c) The field-dependent magnetization curve of FeS nanoplates. (d) T2-weighted MR images of FeS-PEG and iron oxide (Fe3O4) nanoparticles. (e) T2 relaxation rates (R2) of FeS-PEG solutions and iron oxide nanoparticles at different concentrations. The T2 relaxivities (r2) of FeS-PEG and Fe3O4 were determined to be 209.8 mM1 S1 and 99.6 mM1 S1, respectively.

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MR imaging and imaging guided photothermal treatment of cancer. 2. Experimental section

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determine iron levels in various organs. For histological examination, organs from the treated groups and the control group were fixed in 4% formalin and then conducted with paraffin embedded sections for H&E staining. The slices were examined by a digital microscope (Leica QWin).

2.1. Synthesis of FeS nanoplates The FeS nanoplates were prepared by a high temperature chemical synthesis method [41,42]. In brief, 100 mg (0.39 mol) of Iron (II) acetylacetonate (Fe(acac)2), 300 mg (0.78 mol) of trioctylphosphine oxide (TOPO) and 10 ml of oleyamine (OLA) were mixed in 50 ml three-necked flask and degassed at 110  C for 1 h under vacuum. In the presence of nitrogen, the mixture was rapidly heated to 220  C and kept this temperature for 1 h under vigorous magnetic stirring. Afterward, 5 ml OLA solution of sulfur (100 mg) was quickly injected into the solution, which was heated to 220  C and kept for 1 h. After the solution was cooled down to room temperature, a large amount of ethanol was added to precipitate as-grown FeS nanoplates, which were then collected by centrifugation. The yielded FeS nanoplates could be redispersed in cyclohexane for the following experiments. 2.2. Functionalization of FeS nanoplates FeS nanoplates were functionalized through layer-by-layer (LBL) polymer coating method. In brief, 10 mg of FeS nanoplates were re-dispersed in 2 ml of tetrahydrofuran (THF). 40 mg of octylamine-poly (acrylic acid) (OA-PAA) copolymer, which was synthesized following a literature protocol [45], in 2 ml of THF was dropwisely added into FeS nanoplates solution under ultrasonication for 30 min. After stirring for 6 h, we blow-dried the solvent and re-dispersed the OAPAA coated FeS nanoplates in water. Excess OA-PAA was removed by centrifugation at 10,000 rpm for 10 min to discard the supernatant. Next, 40 mg of poly (allylamine hydrochloride) (PAH) dispersed in 2 ml water was dropwisely added into the above solution containing OA-PAA coated FeS under ultrasonication for 30 min. The solution was purified by centrifugation at 10000 rpm for 10 min to remove excess PAH. For the third layer of polymer coating, 40 mg of PAA was added into the above solution under ultrasonication for 30 min. After adjusting the pH to 7.4, 5 mg of EDC was added into the solution to induce cross-linking between PAH and PAA. After stirring for 6 h, the obtained FeS solution was purified by centrifugation to remove excess PAA. Lastly, in order to coat nanoparticles with PEG, 80 mg of 6-armPEG (Mw ¼ 10000) was added into the aforementioned FeS solution. Under ultrasonication, 10 mg of EDC was added into the solution in two equal portions. The reaction was stirred overnight, yielding a FeS-PEG solution, which was purified by filtration through 100 kDa molecular weight cut-off (MWCO) filters to remove excess PEG and stored in 4  C for future experiments. 2.3. Cell and animal models 4T1 murine breast cancer cells and 293T human embryo kidney cells were cultured in standard cell media recommended by American type culture collection (ATCC). Female Balb/C mice were obtained from Nanjing Peng Sheng Biological Technology Co Ltd and used under protocols approved by Soochow University Laboratory Animal Center. The 4T1 tumor models were generated by subcutaneous injection of 2  106 cells in 50 ml PBS into the right abdomen of each female BALB/c mouse. The mice were used for treatment when the tumor volume reached ~100 mm3. 2.4. In vivo tumor MR imaging Mice bearing 4T1 tumors were i.v. injected with FeS-PEG (200 ml, 2 mg/ml) and imaged under 3.0-T animal MRI scanner equipped with a special coil designed for small animal imaging at different time points (Bruker Biospin Corporation, Billerica, MA, USA). 2.5. In vivo photothermal therapy For in vivo photo-thermal treatment, mice bearing 4T1 tumors 24 h post i.v. injection of FeS-PEG (dose ¼ 20 mg/kg) were irradiated with the 808 nm NIR laser (Hi-Tech Optoelectronics Co., Ltd. Beijing, China) at power density of 1 W/cm2 for 5 min. The surface temperature of tumors during laser irradiation was monitored by an IR thermal camera (IRS E50 Pro Thermal Imaging Camera). The tumor sizes were measured by a caliper every other day and calculated as the volume ¼ (tumor length)  (tumor width)2/2. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). 2.6. Biodistribution and toxicology studies Fifteen healthy Balb/c mice were injected with FeS-PEG at the dose of 100 mg/kg and sacrificed at various time points after injection (1, 7 and 50 days, five mice per time point). Another five age-matched healthy Blab/c female mice were scarified as the control group. All the blood parameters were measured in Shanghai Research Center for Biomodel Organisms. Major organs of those mice were harvested and divided into two halves for biodistribution measurement and histological examination, respectively. For biodistribution measurement, major organs, including liver, spleen, kidney, heart, lung, intestine muscle and bone, from mice treated with and without FeS-PEG were solubilized by aqua regia for ICP-AES measurement to

3. Results and discussion Iron sulfide nanoplates were synthesized using trioctylphosphine oxide (TOPO) assisted high temperature hotinjection chemical synthesis method following literature protocols with modifications [41e43]. X-ray powder diffraction (XRD) analysis showed that the chemical structure of the obtained nanoparticles matched well with the standard XRD data of pyrrhotite FeS (JCPDS card, file No. 75-0602) (Supporting information Fig. S1). The XRD results showed that the produced FeS nanoplates exhibited hexagonal structure which has been reported in the previous work by Gao et al. in 2008 [44]. Such hexagonal crystal structure, different from the cubic structure, would lead to the formation of nanoplates instead of spherical nanoparticles. Transmission electron microscopy (TEM) images revealed that the obtained FeS nano-structures were mainly triangle nanoplates with sizes in the range of 32e36 nm (Fig. 1b, Supporting information Fig. S2). High-resolution TEM (HRTEM) imaging of FeS nanoplates gave a lattice spacing of 0.286 nm, which corresponded to the (110) crystal plane (Fig. 1b, inset). In order to offer as-made FeS nanoplates water solubility and enhance their biocompatiblity, FeS nanoplates were functionalized with PEG through a layer-by-layer (LBL) polymer coating method [17,45] (see method section for detailed procedures). An octylamineepoly (acrylic acid) (OAePAA) co-polymer was synthesized [46] and used to functionalize as-made FeS nanoplates which were coated with a layer of hydrophobic TOPO, transferring FeS nanoplates from the oil phase to the aqueous phase. The obtained watersoluble nanoplates with negative charges were then coated subsequently with a cationic polymer poly (allylamine hydrochloride) (PAH), and then an anionic polymer PAA through electrostatic binding together with covalent cross-linking. Lastly, amineterminated six-arm branched PEG (10 kDa) was conjugated to the surface of those nanoparticles via amide formation, obtaining PEGylated FeS nanoplates for our following experiments. The successful LBL polymer coatings were confirmed by zeta potential measurement of those nanoparticles at each step of polymer coating (Supporting Fig. S3a). The final product, PEGylated FeS nanoplates with an average hydrodynamic size at ~120 nm (Supporting information Fig. S3b) exhibited remarkable stability in various physiological solutions including water, saline, cell medium, and serum (Supporting information Fig. S4). It is worth noting that TOPO molecules, which serve as a coating/protecting layer on FeS surface, appears to be critical in maintaining the chemical stability of FeS nanostructures. While our current final product synthesized in the presence of TOPO exhibited great stability in aqueous solutions, FeS nanoplates if fabricated without the addition of TOPO were not stable after being transferred into water, and showed a rapid color change into a yellowish solution, indicating the possible oxidization of FeS (Supporting information Fig. S5). The magnetic property of FeS nanoplates was evaluated by the field-dependent magnetization measurement (Fig. 1c). A tiny hysteresis loop showed up in the S-shape curves, indicating that our FeS nanoplates were mainly superparamagnetic with a small degree of ferromagnetism, in marked contrast to bulk FeS which is known to be antiferromagnetic. We speculated that as the surface/ volume ratio increases, the role of the surface structure in the magnetic behavior becomes more important and determines the superparamagnetic behavior. Comparing with the FeS bulk, the surface structures of FeS nanoplates may be more disordered and

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Fig. 2. Photothermal heating of PEGylated FeS nanoplates. (a) UV-vis-NIR spectra of FeS-PEG and iron oxide nanoparticles at the same concentration (0.05 mg/ml). (b & c) IR thermal images (b) and heating curves (c) of FeS-PEG solutions with different concentrations and an iron oxide solution at 0.5 mg/ml under 808-nm laser irradiation at a power density of 1 W/cm2 for 5 min.

make more noncompensation surface spins. Similar transitions of magnetic behaviors have been observed when other types of bulk antiferromagnetic materials are transformed into nanostructures [48,49].

We thus wondered whether FeS nanoplates could serve as a contrast agent in MR imaging. T2-weighted MR images of FeS-PEG solutions and ultrasmall iron oxide (Fe3O4) nanoparticles (Supporting information Fig. S6) were acquired on a 3.0-T clinical MR scanner,

Fig. 3. In vivo MR imaging. (a) MR images of mice i.v. injected with FeS-PEG (dose ¼ 20 mg/kg) recorded at different time points post-injection. Tumor and liver are pointed with solid and dashed arrows, respectively. (b) The relative T2 signals of tumor and liver sites measured from MR images shown in (a). (c) Prussia blue staining of tumor slices from mice treated with and without FeS-PEG. Blue color, which indicated the existence of Fe, was observed in the tumor of mouse after injection of FeS-PEG. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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revealing the concentration-dependent darkening effect. The transverse relaxivity (r2) of FeS-PEG was measured to be 209.8 mM1 S1, which was higher than that of Fe3O4 (99.6 mM1 S1), as well as several types of Fe-based clinical contrast agents (72 mM1 S1 for ferumoxsil, 151 mM1 S1 for ferrixan, and 98.3 mM1 S1 for ferumoxide) [50] (Fig. 1d & e), promising the use of PEGylated FeS nanoplates as a great T2-MR contrast agent. Different from iron oxide (Fe3O4) nanoparticles with rather low NIR absorbance, PEGylated FeS nanoplates exhibited high absorbance in a wide spectrum range from UV to NIR (Fig. 2a). The weight extinction coefficient of FeS was measured to be 15.5 L g1cm1 at 808-nm, which was lower than those of MoS2 (29.8 L g1cm1), WS2 (23.8 L g1cm1) and reduced graphene oxide (GO) (21.1 L g1cm1), but higher than that of GO (5.94 L g1cm1) and comparable to that of CuS nanoparticles (16.6Lg1cm1) [4,23,29,47]. In order to confirm the potential of FeS-PEG as a photothermal agent, FeS-PEG solutions at different concentrations were irradiated with an 808-nm laser at the power density of 1 W/cm2 for 5 min. Infrared (IR) thermal imaging was used to monitor the temperature changes of those FeS-PEG solutions (Fig. 2b), which showed an obvious concentration-dependent temperature increase under laser irradiation (Fig. 2c), with the highest temperature reached to ~70  C within 5 min. In marked contrast, iron oxide nanoparticles, whose absorbance in the NIR region was low, showed much lower photothermal conversion efficiency even at a high concentration (0.5 mg/ml) (Fig. 2b & c). Therefore, FeS nanoplates appeared to be a rather effective photothermal agent due to their strong NIR absorbance.

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Before any further biological experiments, we firstly tested the potential toxicity of FeS-PEG nanoplates to different cell lines. 4T1 murine breast cancer cells and 293T human embryo kidney cells were selected and incubated with FeS-PEG at different concentrations for 24 h. The standard methyl thiazolyl tetrazolium (MTT) assay was then carried out to determine their relative viabilities. No obvious cytotoxicity of FeS-PEG to these two cell lines was observed even at the highest nanoparticle concentration of 200 mg/ml (Supporting information, Fig. S7a). We then used FeS-PEG as a photothermal agent for in vitro PTT. 4T1 cancer cells were incubated with FeS-PEG at the concentration of 100 mg/ml for 4 h and then irradiated with an 808-nm laser. As the increase of laser power density, the remained cell viabilities showed a remarkable drop as revealed by both MTT assay and Calcine AM & propidium iodide (PI) co-staining assay (Supporting information, Fig. S5b & c). The vast majority of cancer cells were killed after laser irradiation at 1 W/cm2 for 5 min. In contrast, cells without FeS-PEG incubation were not affected even after laser exposure at the highest power density (Supporting information, Fig. S7b & C). Considering the high r2 value of FeS-PEG, we next used T2weighted MR imaging to study the in vivo translocation of PEGylated FeS nanoplates after intravenous (i.v.) injection. Mice bearing 4T1 tumors were i.v. injected with FeS-PEG (200 ml, 2 mg/ml, dose ¼ 20 mg/kg) and imaged by 3.0 T clinical MR scanner equipped with a small animal imaging coil. Remarkable time-dependent darkening effect in the tumor of injected mice was observed (Fig. 3a & b), suggesting the high passive tumor uptake of FeS-PEG nanoplates likely via the enhanced permeability and retention effect of cancerous tumors. The high level of iron was also confirmed

Fig. 4. In vivo photothermal therapy. (a) IR thermal images of tumor-bearing mice with or without FeS-PEG injection under exposure to the 808 nm laser at the power density of 1 W/cm 2 recorded at different time intervals. (b) H&E stained tumor slices collected from FeS-PEG injected mice before and after laser irradiation (1 W/cm2, 5 min). (e & f) The tumor growth curves (c) and survival curves (d) of mice after different treatments, including untreated group (control), laser treated mice without injection of FeS-PEG (laser only), FeS-PEG injected mice without laser irradiation (FeS-PEG only), and FeS-PEG injected mice with laser irradiation (FeS-PEG þ laser). The tumor volumes were normalized to their initial sizes. (e) Representative photos of mice bearing 4T1 tumors taken at day 7 after different treatments indicated.

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by Prussian blue stained tumor slices (Fig. 3c). On the other side, accumulation of nanoplates in reticuloendothelial systems (RES) was also observed as evidenced by the obvious darkening effect in the liver of FeS-PEG injected mice. Note that we were not able to accurately measure the biodistribution of FeS-PEG by the iron concentrations at the imaging & treatment dose (20 mg/kg) due to the high background iron levels in various organs (see data in Fig. 5a). Encouraged by our imaging results, we then studied in vivo cancer PTT using FeS-PEG as a photothermal agent. Mice bearing 4T1 tumors were i.v. injected with FeS-PEG (dose ¼ 20 mg/kg) and irradiated by the 808-nm laser at the power density of 1 W/cm2 for 5 min. An IR camera was used to monitor the surface temperature change of tumors. It was found that the temperature of tumors on mice injected with FeS-PEG rapidly increased to ~60  C within 5 min exposure to the NIR laser. Such temperature would be high enough to ablate tumors in vivo (Fig. 4a). However, in the control group, the surface temperature of tumor without FeS-PEG injection showed no significant increase under laser irradiation at the same conditions (Fig. 4a). Hematoxylin and eosin (H&E) stained tumor slices revealed that tumor cells were severely damaged right after photothermal treatment (Fig. 4b). The therapeutic efficacy of FeS-PEG induced PTT was then carefully evaluated. Four groups of mice bearing 4T1 tumors with 5e7 mice per group were used in our experiments. For the treatment group, seven mice bearing 4T1 tumors were i.v. injected with FeS-PEG (dose ¼ 20 mg/kg). After 24 h, their tumors were irradiated by the 808-nm laser for 5 min (1 W/cm2). The other three control groups included untreated group, mice injected with FeS-PEG but no laser irradiation, and mice exposed to the laser but without FeS-

PEG injection. The tumor sizes were measured by a caliper every the other day after treatment. The tumors in all three control groups showed the similar growth speed, suggesting that neither laser irradiation of tumors nor FeS-PEG injection alone would affect the tumor growth (Fig. 4c & e). In marked contrast, tumors of mice injected with FeS-PEG and after laser exposure were completely ablated after PTT treatment, without showing a single case of regrowth within 60 days (Fig. 4d). Therefore, our synthesized FeSPEG could serve as a powerful photothermal agent for in vivo cancer ablation. Finally, to ensure the safe use of FeS-PEG, healthy Balb/c mice were i.v. injected with an ultra-high dose of FeS-PEG at 100 mg/kg, which was 5 times of the treatment dose, and scarified at 1, 7 and 50 days post-injection (p.i.) for long-term biodistribution measurements and toxicology examinations (5 mice per group). Another group of 5 healthy mice without FeS-PEG injection were scarified at day 50 as the control. For biodistribution study, all collected organs from mice treated with and without FeS-PEG were solubilized by aqua regia for inductively coupled plasma atomic emission spectrometry (ICP-AES) measurement of iron concentrations. As expected and consistent to MR imaging results, high levels of iron contents were observed in RES organs such as liver and spleen owing to the macrophages uptake of nanoparticles (Fig. 5a). Interestingly, over time we observed a persistent decrease of iron levels in all measured major organs. After 50 days, the iron levels in mice after injection with such a high dose (100 mg/kg) of FeS-PEG dropped back to normal levels in most of studied organs except spleen, in which the iron content was still 2 times of the untreated control. Prussia blue staining of liver and spleen slices also evidenced the gradual clearance of iron from mice i.v. injected with

Fig. 5. In vivo long-term biodistribution of FeS-PEG. (a) Time-dependent biodistribution measurement of Fe levels in various organs of mice after i.v. injection of FeS-PEG at an ultrahigh dose of 100 mg/kg. (b) Prussia blue stained micrographs of spleen and liver slices from FeS-PEG treated mice taken at different time points (1, 7 and 50 days p.i.).

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Fig. 6. In vivo potential toxicity of FeS-PEG. (aed) Blood biochemistry of mice treated with FeS-PEG at dose of 100 mg/kg measured at 1, 7 and 50 days p.i: (a) The blood level of ALP, AST and ALT from the mice treated with and without FeS-PEG. (b) BUN level in the blood. (c) Albumin/globin (A/G) rations in the blood. (d) ALB level in the blood. (eel) The blood routine analysis from healthy control and treated mice: white blood cells (e), hemoglobin (f), hematocrit (g), platelets (h), red blood cells (i), mean corpuscular hemoglobin (j), mean corpuscular volume (k), and mean corpuscular hemoglobin concentration (l). All the parameters of blood analysis fell well in the respective normal ranges.

FeS-PEG (Fig. 5b). We speculate that FeS nanoplates in the complicated in vivo environment may be gradually decomposed into Fe ions, which could then be excreted from the treated animals. Although we did not notice any short-term side effects to the treated mice, potential long-term toxicity of those FeS-PEG nanoplates remained a concern. During our experiments, we did not observe any death or significant body weight drop for FeS-PEG injected mice at the dose of 100 mg/kg within 50 days (data not shown). Blood analysis including blood chemistry and blood routine examination was carried out at different time points postinjection of FeS-PEG. In blood chemistry analysis, the liver function markers including alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and serum albumin (ALB), kidney function marker urea nitrogen (BUN), as well as albumin/globin ration, were all measured to be normal (Fig. 6aed), indicating no obvious hepatic and kidney disorder of mice induced by FeS-PEG even at high dose of 100 mg/kg. In blood routine examination, we selected white blood cells, red blood cells, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean

corpuscular hemoglobin and platelet count as main parameters to evaluate the possible toxicity of FeS-PEG to mice. The results showed that all of the above parameters in the FeS-PEG treated groups at different time p.i. appeared to be normal compared with those in the control group (Fig. 6eel). Histological examination was also conducted by H&E staining of major organ slices including liver, spleen, kidney, heart and lung (Supporting information, Fig. S8). No notable lesion, inflammation, or other abnormality was observed for the main organs that we examined at various time points after injection of FeS-PEG (Supporting information, Fig. S8). Therefore, FeS-PEG appeared to be a rather safe nano-agent with inducing any obvious side effect to the treated mice even at a very high dose. 4. Conclusions We have for the first time developed PEGylated FeS nanoplates with a high r2 relaxivity and strong NIR-absorbance as a novel multifunctional nano-agent for in vivo MR imaging guided photothermal cancer treatment, achieving excellent therapeutic effect in

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our mouse tumor model experiments. Importantly, it has been found that PEGylated FeS nanoplates after systemic administration could be gradually excreted from major organs of mice, and showed no appreciable toxicity to the treated animals even at an ultra-high dose (100 mg/kg), which was 5 times as high as the imaging & treatment dose. Compared with conventional photothermal agents such as ‘nano-carbons’ and ‘nano-golds’, as well as recently reported NIR-absorbing metal sulfide nanomaterials including CuSx, MoS2, and WS2, FeS nanoplates presented in this work with intrinsic magnetic properties provide unique advantages in imaging-guided therapy. Although the mass extinction coefficient of FeS is slightly lower than that of MoS2, WS2 and reduced graphene oxide, it is comparable to that of CuS nanoparticles and higher than that of graphene oxide, and could still offer great photothermal therapeutic effect as demonstrated in our animal experiments. More importantly, both iron and sulfur elements are quite abundant in lives. Our PEGylated FeS nanoplates have been found to be rather safe to mice at a very high dose. Considering the fact that iron oxide nanoparticles have also been used in the clinic for many years, it is reasonable to believe that iron sulfide nanoagents with appropriate surface coatings may indeed have a considerable chance to enter the clinic, for applications in MR imaging as well as cancer theranostics. Acknowledgments This work was partially supported by a research start-up fund of Soochow University and Research Grants Council of Hong Kong SAR e CRF Grant (Grant No. CityU5/CRF/08). We thank Dr. Haizhen Deng for her great This work was partially supported by the National Natural Science Foundation of China (51302180, 51222203, 51132006, 21207164, 31400861, 81471716), the National “973” Program of China (2011CB911002, 2012CB932601, 2014CB931900), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.10.052. References [1] Yang K, Hu L, Ma X, Ye S, Cheng L, Shi X, et al. Multimodal imagingguided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles. Adv Mater 2012;24:1868e72. [2] Yang Y, Shao Q, Deng R, Wang C, Teng X, Cheng K, et al. In vitro and in vivo uncaging and bioluminescence imaging by using photocaged upconversion nanoparticles. Angew Chem Int Ed 2012;51:3125e9. [3] Yang K, Wan J, Zhang S, Tian B, Zhang YJ, Liu Z. The influence of surface chemistry and particle size of nanoscale graphene oxide on photothermal therapy of cancer using ultra-low laser power. Biomatreials 2012;33: 2206e14. [4] Yang K, Zhang S, Zhang G, Sun X, Lee ST, Liu Z. Graphene in mice: ultra-high in vivo tumor uptake and photothermal therapy. Nano Lett 2010;10:3318e23. [5] Lal S, Clare SE, Halas NJ. Nanoshell-enabled photothermal cancer therapy: impending clinical impact. Acc Chem Res 2008;41:1842e51. [6] Liu HY, Chen D, Li LL, Liu TL, Tan LF, Wu XL, et al. Multifunctional gold nanoshells on silica nanorattles: a platform for the combination of photothermal therapy and chemotherapy with low systemic toxicity. Angew Chem Int Ed 2011;50:891e5. [7] Song X, Gong H, Yin S, Cheng L, Wang C, Li Z, et al. Ultra- small iron oxide doped polypyrrole nanoparticles for in vivo multimodal imaging guided photothermal therapy. Adv Funct Mater 2014;24:1194e201. [8] Chuang YC, Lin CJ, Lo SF, Wang JL, Tzou SC, Yuan SS, et al. Dual functional AuNRs@MnMEIOs nanoclusters for magnetic resonance imaging and photothermal therapy. Biomaterials 2014;35:4678e87. [9] Liu X, Tao H, Yang K, Zhang S, Lee ST, Liu Z. Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors. Biomaterials 2011;32:144e51.

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FeS nanoplates as a multifunctional nano-theranostic for magnetic resonance imaging guided photothermal therapy.

In this work, we develop magnetic iron sulfide (FeS) nanoplates as a theranostic agent for magnetic resonance (MR) imaging-guided photothermal therapy...
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