Fe3O4-Based Multifunctional Nanospheres for Amplified Magnetic

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FeO-Based Multifunctional Nanospheres for Amplified Magnetic Targeting Photothermal Therapy and Fenton Reaction Huanhuan Wu, Keman Cheng, Yuan He, Ziyang Li, Huiling Su, Xiuming Zhang, Yanan Sun, Wei Shi, and Dongtao Ge ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00468 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 2018

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ACS Biomaterials Science & Engineering

Fe3O4-Based Amplified

Multifunctional Magnetic

Nanospheres

Targeting

for

Photothermal

Therapy and Fenton Reaction Huanhuan Wu,#† Keman Cheng,#† Yuan He,† Ziyang Li,† Huiling Su,† Xiuming Zhang,† Yanan Sun,† Wei Shi,†* and Dongtao Ge†* †Department of Biomaterials, Key Laboratory of Biomedical Engineering of Fujian Province university, Research Center of Biomedical Engineering of Xiamen, College of Materials, Xiamen University, Xiamen 361005, Fujian, China Corresponding Author: E-mail: [email protected]; [email protected] Address: Department of Biomaterials, Xiamen University, 422 Siming South Road, Xiamen 361005, China (W.S., D.G.) KEYWORDS: iron oxide, polypyrrole, Fenton Reaction, photothermal therapy, magnetic targeting

ACS Paragon Plus Environment

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ABSTRACT. Multifunctional nanoplatforms have attracted the interests of many scientists because they can achieve better therapeutic effect in the combined treatment of cancer. A novel cancer therapeutic strategy which combining Fe3O4-based in vivo Fenton reaction with polypyrrole (PPy)-based photothermal therapy (PTT) was proposed. The multifunctional nanocomposite was comprised of Fe3O4 as core, PPy as shell and polyethylene glycol. PPy could absorb near-infrared (NIR) light strongly and convert it into heat for tumor photothermal ablation and Fe3O4 NPs were used as a target component to guide the nanoparticles to the tumor site under external magnetic field. It was found that the PPy coating could be used not only for inducing PTT to ablate tumor cells, but also promoting Fe2+/3+ release from Fe3O4 nanoparticles. In vitro cell experiments confirmed that the increased Fe2+/3+ release could effectively enhance the Fe3O4-based Fenton reaction which catalyzed the conversion of H2O2 into highly toxic hydroxyl radical (·OH), thus inducing tumor cell apoptosis. Furthermore, our experiments also showed that the PPy coating could generate photothermal effect to kill 4T1 tumor cells under NIR light exposing but did no harm to normal cells in the dark. Under the guidance of the magnet, we found Fe3O4@PPy-PEG (Fe3O4@P-P) nanoparticles could effectively enrich in tumor site and the therapeutic effect from PTT and photothermal strengthened Fenton reaction was also verified in vivo. It is confirmed for the first time that the photothermal effect could promote the release of iron ions from Fe3O4 at acid condition and enhance the Fenton reaction. Therefore, the Fe3O4@P-P nanoparticles, combined with Fenton reaction and photothermal effect, and obviously the magnetic targeting and magnetic resonance imaging ability, are able to be a candidate for novel tumor theranostic agents.


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INTRODUCTION Cancer is still one of the most intractable diseases in the world.1 With the development of technology and medical science, anticancer therapies, such as chemotherapy,2 radiotherapy3 and surgical intervention, remain mainstay treatments for various types of cancers. These therapies are not entirely specific to tumor cells, they are harmful to healthy organ or normal cells and generate severe side effect to the patients.4,5 Therefore, it is in urgent need of an efficient and low-toxic strategy to improve therapeutic outcomes. Reactive oxygen species (ROS)-mediated chemotherapy, as a novel strategy, has demonstrated outstanding efficacy against cancer cells.6,7 Reactive oxygen species include hydrogen peroxide, superoxide anion radicals, hydroxyl radicals and singlet oxygen.6 Intracellular ROS can be maintained within a specific physiological range by endogenous regulation.8-12 However, overproduction of ROS by exogenous control could disturb the homeostasis ， resulting in oxidation of proteins and nucleic acids, lipid peroxidation and DNA damage.13-16 Fenton reaction as an anticancer strategy which can promote the production of reactive oxygen species has been studied widely. Fenton reaction exploits Fe2+ or Fe3+, acting as a catalyst, to convert hydrogen peroxide to highly toxic ·OH radicals.16 The function of hydroxyl radical is to oxidize biomolecules, leading to cell apoptotsis.17 Therefore, hydroxyl radicals with strong oxidizing as a cancer therapeutic strategy have been extensively studied. It has been confirmed that hydrogen peroxide in tumor is higher than normal organs.18,19 Accumulation of Fe2+/3+ at tumor site becomes a key issue for the initiation of Fenton reaction. Iron oxide nanoparticles have been widely used in biomedical fields, such as magnetic resonance imaging (MRI),20 biomolecular separations21 and magnetic targeting drug delivery.22,23 Considering the fact that the Fe3O4 nanoparticles can release iron ions under certain conditions, it is reasonable to postulate that


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Fe3O4 may be used for the catalytic Fenton reaction to disrupt redox homeostasis in tumor cells, resulting in severe damage and leading to programmed cell death. However, the amount of Fe2+/3+ released from Fe3O4 is usually too low to initiate Fenton reaction, resulting little ROS generation and not significant damages to tumor cells. Local delivery of H2O2 is an efficient method to enhance Fenton reaction,24,25 but more H2O2 in vivo may cause severe damage to normal organ. Therefore, it is an urgent need for a strategy to improve the release of Fe2+/3+ from Fe3O4. On the other hand, nanomaterials with photothermal effect have attracted much attention due to their inherent properties.26-29 Photothermal therapy has been developed as an encouraging cancer therapy which utilizes near-infrared (NIR) light-absorbing nanomaterials to convert optical energy to heat to “cook” tumors. The most-widely studied PTT nanomaterials including gold nanomaterials,30 carbon nanomaterials,31 Pd nanosheets32 and copper sulfide.33 Polypyrrole (PPy) nanomaterials with high conductivity, broad and strong NIR absorption, high photothermal conversion efficiency and good biocompatibility have been widely investigated for photothermal therapy34-36, bioelectronics37,38 and tumor imaging39. We have detailed compared PPy with the previously works in Table S1. The synthesis methods of multifunctional nanocomposites consisting of magnetic nanomaterials (including Fe3O4) and PPy have been developed.40-45 Although Fe3O4@PPy nanocomposites could be successfully synthesized by a variety of methods, Fenton reaction based on Fe3O4 was not studied before. In the present work, a multifunctional nanosphere comprised of Fe3O4 as core, PPy as shell and polyethylene glycol was prepared for tumor combination therapy. It was found that the photothermal effect offered by laser irradiated PPy coating could not only be used to ablate cancer cells, but also enhance the release of Fe2+/3+ from Fe3O4 nanoparticles. More Fe2+/3+


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release could effectively enhance the Fe3O4-based Fenton reaction to provide adequate ROS to induce tumor cell apoptosis. After being modified by PEG, the nanoparticle shows good stability in vivo and prolongs its blood circulation time. Therefore, the Fe3O4@PPy-PEG (abbreviated to Fe3O4@P-P in the subsequent article) nanoparticles combined Fenton reaction and PTT for treating tumor, finally realizing good combination therapeutic effect. Furthermore, Fe3O4 was also used for MRI and magnetic targeting. The in vitro and in vivo results indicated that the excellent combination therapeutic efficacy was achieved. Our results show that the use of Fe3O4@P-P nanoparticles can successfully combine Fenton reaction with photothermal therapy and provide a novel strategy for offering Fe3O4-based nanoparticles Fenton reaction ability for cancer therapy. MATERIALS AND METHODS Materials. Ferric chloride (FeCl3·6H2O), diethylene glycol (DEG), trisodium citrate (Na3Cit), anhydrous sodium acetate (NaOAc), dimethylsulfoxide (DMSO) and Pyrrole monomer (98%) were bought from Sinopharm Chemical Reagent Co. Ltd. (China). Poly(vinyl alcohol) (PVA, MW 9000-10000), dodecylbenzenesulfonic acid sodium salt (SDBS), triethylamine (TEA), 1ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and dichloromethane

were purchased

from Sigma-Aldrich. Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Fetal bovine serum (FBS), and trypsin were bought from Biological Industries (Beit Ahemeq, Israel). Annexin V-FITC, propidium iodide (PI), 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), and calcein-acetoxymethyl (calcein-AM) were bought from KeyGen Biotech Co. Ltd. (China). 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was purchased from Beyotime® Biotechnology (Shanghai, China). NAcetyl-L-cysteine (NAC) were obtained from Aladdin.


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Synthesis of Fe3O4 Nanoparticles. The synthetic method of Fe3O4@P-P was briefly introduced in Scheme 1. First, Fe3O4 NPs were fabricated by solvothermal method according to the verified protocol.46 Briefly, 20 mL of diethylene glycol (DEG) was mixed with 2 mmol FeCl3·6H2O to form a solution. Then, 0.8 mmol trisodium citrate (Na3Cit) was added to the above solution, magnetic stirred at 80 ℃ until completely dissolved. 6 mmol NaOAc was dropwise added into the above mixture with magnetic stirring for 30 min. Following this, a Teflon-lined stainless-steel autoclave was then used for containing the mixed solution and sealed in air. The autoclave was heated at 240 °C for 6 h. After the autoclave temperature restores up to room temperature naturally, Fe3O4 nanoparticles were separated by centrifugation at 12000 rpm for 20 min, washed with ethanol and water several times. The obtained precipitate was finally dried at 60 ℃ under vacuum for 12 h. Preparation of Fe3O4@PPy Nanoparticles. An in situ chemical oxidative polymerization was utilized to synthesize the Fe3O4@PPy nanoparticles. 10 mg Fe3O4 nanoparticles were well dispersed in a mixed solution comprised of 30 mg PVA and 10 mg SDBS. The above solution was ultrasonicated (100 W) for over 40 min. The obtained mixture was mechanical stirred for 2 h under room temperature and then 40 μL of pyrrole was introduced into the solution, then the reaction mixture was stirred for 30 min. After that, 40 mg of FeCl3·6H2O at 1 mL deionized water was added into the above solution. Finally, the solution was further stirred at room temperature for 12 h. The resulting nanoparticles were carefully collected and centrifuged at 16000 rpm for 15 min. Finally, deionized water was used to wash the obtained Fe3O4@PPy nanoparticles for several times and magnets were used to remove excess reagents and PPy. Synthesis of Fe3O4@P-P Nanospheres. Preparation of C18PMH-PEG was conducted on a previously published method.47 Briefly, 143 mg mPEG-NH2 (5K) and 10 mg C18PMH were


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dissolved in a mixed solution which was comprised of 6 μL TEA and 5 mL dichloromethane. After adding 11 mg EDC to the reaction solution, the mixture was further stirred for 24 h. After that, the solvent was evaporated and dialyzed to remove the unreacted reagent. C18PMH-PEG was further freeze-dried and kept at -20 ℃ for further use. As for the preparation of Fe3O4@P-P nanoparticles, the synthesized C18PMH-PEG (2 mg) was well dispersed in 1 mL deionized water under ultrasonication (100 W) for 2 min. After dropwisely adding the C18PMH-PEG solution to the Fe3O4@PPy solution, the mixture was ultrasonicated for another 30 min. Finally, the product was purified and washed with water by centrifugation for several times. Characterization. Fe3O4 (1 mg), Fe3O4@PPy (1 mg) and Fe3O4@P-P (1 mg) were dissolved in 1 mL of PBS (pH 7.4) under ultrasonication (100 W) for 2 min. The morphology of Fe3O4@PPy and Fe3O4@P-P nanoparticles were monitored by transmission electron microscopy (TEM, JEOL JEM-2100, Japan). The hydrodynamic diameters and surface zeta potential of the nanoparticles were carried out on a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.). UV-vis absorption spectra were obtained by a UV-vis spectrophotometer (UV-1750; Shimadzu, Japan). The concentration of iron ions of different nanoparticles was measured by inductively coupled plasma-mass spectrometry (ICP-MS, Perkin-Elmer). The powder of Fe3O4@PPy (1 mg) and Fe3O4@P-P (1 mg) was analyzed by Fourier transform infrared (FT-IR) (Thermo Scientific) and X-ray diffraction (Shimasz Cu Kα radiation (λ = 1.54060 Å), 30 kV, 30 mA). Field-dependent magnetization (M-H) was detected by VSM-7400 magnetometer (Lakeshore, America). Photothermal Effect of Fe3O4@P-P. Fe3O4@P-P nanoparticles with different mass concentrations (0, 25, 50, 75,100,150 μg/mL) were suspended in quartz cuvettes, then put them


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under NIR laser irradiation (808 nm, 1 W/cm2) for 10 min. Fe3O4 (150 μg/mL) and Fe3O4@PPy nanoparticles (150 μg/mL) were also irradiated by the same exposure conditions as negative or positive control, respectively. An IR thermal imager (FOTRIC-225) was used to obtain the thermal images of the solutions. In addition, the photostability of Fe3O4@P-P nanoparticles was also studied by laser irradiation for 5 minutes, cooling to room temperature, and then irradiation for another 5 min. This cycle was repeated for four times. Iron Release Behavior of Fe3O4@P-P. Fe3O4@P-P nanoparticles were dispersed in 1 mL PBS buffered solution (1 mM) with different pH (pH=7.4 and 5.0). The laser irradiation groups were further exposed to NIR laser irradiation (808 nm, 1 W/cm2) for 10 min. Then, the samples were transferred into in a dialysis bags (1000 Da) and the bags were immersed in 20 mL PBS at pH 7.4 or 5.0. The dialysis process was carried out in an oscillating chamber (37 ℃, 200 rpm). After collecting 2 mL solution outside the dialysis bag at certain time points, the same amount of new solution was added. The release amount of iron ions was detected by ICP-MS. The effect of pH and temperature on the release of iron ions from Fe3O4 nanoparticles were then studied. We performed a TEM analysis and detected the size of the Fe3O4 nanoparticles at different conditions. Briefly, 2.4 mg Fe3O4 nanoparticles were dissolved in 4 mL PBS and the pH of the solution was adjusted to 7.4. The solution was divided equally into four groups. The groups were adjusted to various conditions (pH 7.4, pH 7.4+60 ℃, pH 5.0 and pH 5.0+60 ℃). After incubation for 5 min, the samples were characterized by dynamic light scattering (DLS) and TEM. Analysis of ·OH Production by Methylene Blue (MB). MB could be degraded by ·OH and weaken the absorption of MB at 664nm.48 2 mL water solution containing H2O2 (0.1 mM, pH 5.0), MB solution (10 mg/L, pH5.0) and Fe3O4 (100 μg/mL) were prepared as the so divided


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groups. Prior to reaction, the reaction solution was treated with ultrasonication for 5 min and then the above solutions were kept at room temperature or 60 ℃ for 20 min. Finally, UV-vis spectrometer was used to detect the absorbance of the reaction solution at 664 nm. In Vitro MR Imaging. 7.0-T small-animal MR system (Bruker biospin, Germany) was utilized to calculate the T2 relaxivity of Fe3O4@PPy or Fe3O4@P-P NPs. The Fe3O4@PPy or Fe3O4@P-P NPs were dissolved in pure water with different iron concentrations (0-0.5 mM). The MR images of the above solutions were obtained by the T2-TurboRARE sequence ((rapid acquisition with relaxation enhancement): TR (repetition time)/TE (echo time) = 3000.0/40.0 ms, slice thickness = 1 mm, average times = 16, FOV (field of view) = 4 cm × 4 cm, matrix = 256 × 256). Finally, T2 relaxation rates were plotted against the Fe concentrations. Cell Lines and Animal Models. Cells lines used in our experiments were Human umbilical vein endothelial cells (HUVEC) and 4T1 murine breast cancer cells (4T1). They were supplied by the American Type Culture Collection and cultured in a cell incubator (37 °C, 5% CO2). The cells were cultured in DMEM (HUVEC)/1640 (4T1) medium supplemented with 10% FBS and 1% streptomycin/penicillin. Female Balb/c mice were obtained from Laboratory Animal Center of Xiamen University. All animal experiments were performed under the guidelines of the Institutional Animal Care and Use Committee. 2×106 4T1 tumor cells were subcutaneously inoculated into the back of female Balb/c mice for obtaining the 4T1 tumor models. Cell Uptake Efficiency of Fe3O4@P-P Nanoparticles. 1×104 4T1 tumor cells were incubated in RPMI 1640 medium (500 μL) containing varied concentration of Fe3O4@P-P nanoparticles (25-150 μg/mL) in the cell incubator (37 °C, 5% CO2). After incubating for 24 h, PBS was used to wash the cells for five times. Then, 4T1 cells were further treated with 37% HCl. The Fe content was quantified by ICP-MS to reflect the content of Fe3O4@P-P nanoparticles in cells.


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To further evaluate the time dependent cell uptake of Fe3O4@P-P nanoparticles in vitro, 1×104 4T1 tumor cells were cultured in RPMI 1640 medium (500 μL) and then 100 μg/mL Fe3O4@P-P nanoparticles were added at 1 h, 3 h, 6 h, 12 h, 18 h and 24 h. After incubation, the cells were treated as mentioned before, and the Fe3O4@P-P nanoparticles in cells were detected by quantifying the Fe content with ICP-MS. MTT Assay. The cell viability of 4T1 and HUVEC cultured with Fe3O4@P-P nanoparticles were analyzed by MTT assay. Briefly, HUVEC and 4T1 cells were preseeded in 96-well cell culture plates (1 × 105 cells per well). After incubated in a cell incubator (37 °C, 5% CO2) for 12 h, different concentrations (0, 25, 50, 75, 100, 150 μg/mL) of Fe3O4@P-P nanoparticles dispersed in fresh medium were introduced to the cells. Then, the above cells were incubated for another 24 h. After adding PBS to wash the cells for three times, MTT (10 μL, 5 mg/mL) was added into each well. After another 4 h incubation, the MTT solution was carefully removed and DMSO (150 µL per well) was added to dissolve the formazan crystals. After shaken gently for 10 min, the absorbance at 570 nm was detected by a microplate reader. In Vitro Photothermal Therapy Effect. 4T1 cells in 96-well plates (1×105 cells per well) were cultured in a cell incubator (37 °C, 5% CO2) for 12 h. Then, different concentrations of nanoparticles were added to the cells. After another 12 h incubation, NIR laser (808 nm,1 W/cm2) was used to irradiate the cells for 5 min. Finally, the cell viability was determined by the standard MTT assay. As for the staining of live and dead cells, 4T1 cells in 12-well plates (1×105 cells per well) were cultured in a cell incubator (37 °C, 5% CO2) for 12 h. Then, the cells treated with PBS or PBS containing 0.1 mM H2O2 or 100 μg/mL Fe3O4@P-P nanoparticles for 2 h. Then, NIR laser (808 nm, 1 W·cm−2) was used to irradiate the groups needed laser for 5 min. After the old


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medium was removed and the cells were washed with PBS for three times, the fresh medium containing calcein-AM and PI were added into the cells for staining. The fluorescence photos of the samples were obtained with a fluorescent microscopy (DM600B; LEIKA). Cell Apoptosis. 4T1 cells were seeded in a 6-well plate (5×104 cells per well). After 12 h incubation, the cells were divided into different groups and treated with PBS or PBS containing 0.1 mM H2O2 or 100 μg/mL Fe3O4@P-P nanoparticles for 2 h. After that, NIR laser (808 nm, 1 W·cm−2) was introduced to irradiate some groups for 5 min. Finally, all cells containing in suspension or culture dish were carefully collected for further analysis. The staining process was conducted by the protocol of FITC Annexin V Apoptosis Detection Kit. Briefly, 100 μL 1X binding buffer was used to resuspend cells (1×105). Afterwards, 5 µL PI and 5 µL FITC Annexin V were introduced into cell suspension. After gently vortex, the cells were incubated at room temperature for another 15 min in the dark. Finally, another 400 µL 1X binding buffer was added into each group. The suspension was analyzed by flow cytometry within 1 h. Investigation of Intracellular ROS Level In Vitro. Reactive Oxygen Species Assay Kit (Beyotime Biotechnology) was used to analyze the intracellular ROS level through observing the fluorescence intensity of DCF (generated by DCFH-DA decomposition).49 Briefly, 4T1 tumor cells were seeded in a 6-well cell culture plate before the experiment. 0.1 mM H2O2 was added to the cells for simulating tumor microenvironment. Then, cells were divided into five groups including PBS group was used as control, PBS+L (PBS + Laser irradiation); Fe3O4@P-P+N+L (Fe3O4@P-P nanoparticles + NAC + Laser irradiation); Fe3O4@P-P+L (Fe3O4@P-P nanoparticles + Laser irradiation). 5 mM ROS scavenger N-acetyl-L-cysteine (NAC) was used in this experiment. NIR laser (808 nm, 1 W·cm−2) was used to irradiate the groups that need laser for 5 min. After 24 h incubation, cells were transferred to 1.5 mL centrifuge tube. 200 μL DCFH-


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DA (10 mM) was used to resuspend the collected cells. After incubated for 30 min at 37 ℃, cells were collected by centrifugation (1000 rpm, 5 min). Before the flow cytometric analysis, PBS was used to clean the cells for three times. A total of 10000 events were analyzed for each sample. For confocal microscopy analysis, 4T1 tumor cells were seeded onto the 35 mm CLSMexclusive culture disk. When the cells were 80% confluent, they were incubated with 0.1 mM H2O2. Then, cells were divided into five groups as mentioned before. After 24 h incubation, cells were cleaned with PBS for three times. Then cells were incubated with DCFH-DA in the dark at 37 ℃ for 30 min. Finally, PBS was used to clean cells for three times, the live cells were observed under the ZEISS LSM710 confocal microscopy. In Vivo Biodistribution Evaluation of Fe3O4@P-P. We utilized ICP-MS to evaluate the biodistribution of the Fe3O4@P-P NPs in 4T1 tumor-bearing Balb/c mice (female, 6 weeks years old). The mice were divided into three groups (Saline, Fe3O4@P-P NPs and Fe3O4@P-P NPs with magnet). Fe3O4@P-P NPs (100 mg/kg body weight) were intravenously injected into the mice. The group that need magnetic field was provided by NdFeB magnets and the magnets were stuck on the surface of tumor with double-sided glue and wrapped by cloth. At the certain time intervals, the mice were sacrificed. The organs were separated, weighted and digested according to the previous method51. Before the ICP-MS analysis, different concentrations (0, 5, 10, 50, and 100 ppb) of the Fe standard solutions were dissolved in the 2 % HNO3 solution. The prepared organs solutions and standard solutions were analyzed by ICP-MS. The content of Fe3O4@P-P NPs was eventually normalized to the weight of tissue per gram. The mice injected with saline were acted as control groups.


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In Vivo MR Imaging of Fe3O4@P-P NPs. 4T1 tumor-bearing nude mice (n = 3) with tumor size about 300 mm3 were used for in vivo MR imaging. 100 µL of Fe3O4@P-P NPs were intravenously injected into mice with and without magnetic field for 30 min. The mice treated with saline were regarded as control. Magnetic field was produced by NdFeB magnets (a cylinder with a thickness of 1.5 mm and a diameter of 8 mm) and we put magnets on the tumor of tumor-bearing mice with double-sided glue and wrapped the magnets in cloth. Thereafter, in vivo T2-weighted MR images were obtained by a T2-weighted sequence (MESE), TR/TE = 3000.0/40.0 ms, slice thickness = 1.0 mm, average times = 25, FOV = 3.0 cm × 3.0 cm, matrix = 256 × 256. In Vivo Antitumor Therapy. 4T1 tumor-bearing Balb/c mice (with tumor size about 200 mm3) were divided into 7 groups on average (4 mice per group) including Saine group which was used as negative control, Saline+L (Saline + Laser irradiation); NPs (Fe3O4@P-P nanoparticles); NPs+M (Fe3O4@P-P nanoparticles + magnet); NPs+L (Fe3O4@P-P nanoparticles + Laser irradiation); NPs+M+N+L (Fe3O4@P-P nanoparticles + Magnet + NAC + Laser irradiation); NPs+M+L (Fe3O4@P-P nanoparticles + Magnet + Laser irradiation). Then, different formulations were intravenously injected into the mice at a Fe dose of 3.3 mg (kg)-1 body weight. NAC (20 mg (kg)-1 body weight) was intratumorally injected into mice. NIR laser (808 nm, 1 W·cm−2) was used to irradiate the groups that need laser for 5 min once a week. Magnetic field was also produced by NdFeB magnets (a cylinder with a thickness of 1.5 mm and a diameter of 8 mm) and magnets were stuck on the surface of tumor with double-sided glue and wrapped by cloth. The temperature change at the tumor region and thermal imaging were captured by IR thermal camera. After that, tumor volumes were studied at various times and recorded every


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other day. The tumor volumes of mice were calculated according to the following formula: Volume = width2 × length × 0.5. The body weight of the mice was recorded every other day. After treated with different formulations, the mice were euthanized. The major organs (heart, liver, spleen, lung, kidney) and tumor were separated and stored in 4% paraformaldehyde for further histological analysis by hematoxylin and eosin (H&E) staining. Detection of ROS Generation In Vivo. To evaluate the ROS induced by NPs within tumors, tumors of different groups were collected and stained with DCFH-DA according to the manufacture’s protocol. Briefly, the tumor tissues were cut into small pieces and placed in PBS (Ph 7.4) containing 2% fetal bovine serum. The above solution was then transferred to a glass homogenizer, and then the single cells suspension was prepared under mild pressure without digestive enzymes. Red blood cells were removed by adding RBC lysis buffer. Finally, PBS containing 10 mM DCFH-DA was used to stain cells and the level of ROS in tumors were evaluated by flow cytometry. Statistical Analysis. SPSS 17.0 statistical analysis software was used for Analysis. Statistical analysis was conducted by the Student t test for comparison of two groups, followed by Newman–Keuls test, *p

Fe3O4-Based Multifunctional Nanospheres for Amplified Magnetic

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