External Magnetic Field-Enhanced Chemo-Photothermal Combination

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External Magnetic Field Enhanced Chemo-Photothermal Combination Tumor Therapy via Iron Oxide Nanoparticles Xiaomeng Guo, Wei Li, Lihua Luo, Zuhua Wang, Qingpo Li, Fenfen Kong, Hanbo Zhang, Jie Yang, Chunqi Zhu, Yongzhong Du, and Jian You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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ACS Applied Materials & Interfaces

External Magnetic Field Enhanced Chemo-Photothermal Combination Tumor Therapy via Iron Oxide Nanoparticles

Xiaomeng Guo, Wei Li, Lihua Luo, Zuhua Wang, Qingpo Li, Fenfen Kong, Hanbo Zhang, Jie Yang, Chunqi Zhu, Yongzhong Du, and Jian You*

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, P. R. China

* Corresponding Author: Jian You, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, Zhejiang, China. Office: 086-571-88981651 Fax: 086-571-88208439 Email: [email protected].

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Abstract The development of multifunctional nanoplatforms based on magnetic nanoparticles (MNPs) has attracted increasing attention. MNPs especially exhibit excellent responsiveness under the guidance of an external magnetic field (MF), resulting in tumor-specific, targeted delivery. The behavior and magnetic-targeting efficiency of MNPs largely depend on their physiochemical properties, especially the particle size; however, the optimal size range may vary across the multiple bioapplications associated with multifunctional nanoparticles. The optimal size range of nanoparticles for external MF-mediated targeted delivery has rarely been reported. In this work, we synthesized a series of monodisperse Fe3O4 nanoparticles with identical surface properties ranging in size from 10 to 310 nm, and we systematically investigated their behavior and MF-assisted antitumor efficacy. Our data indicated that smaller Fe3O4 nanoparticles exhibited greater cellular internalization, while larger Fe3O4 nanoparticles showed greater tumor accumulation. Larger Fe3O4 nanoparticles exhibited stronger magnetic responsiveness both in vitro and in vivo, which could be used to further induce increased accumulation of nanoparticles and their payload (e.g., doxorubicin) into the tumor site under the guidance of an external MF. Our work demonstrated that larger Fe3O4 nanoparticles, with a diameter of up to 310 nm, exhibited the best magnetic-targeting efficiency mediated by an external MF and the strongest antitumor efficacy from combination photothermal-chemotherapy. Our results could serve as a valuable reference for the future design of MNPs and their targeted delivery via the modulation of an external MF.

KEYWORDS External magnetic field; iron oxide nanoparticles; magnetic responsiveness; tumor targeting; chemotherapy


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Introduction The efficient delivery and specific targeting of theranostic agents to a tumor region are of the utmost importance in cancer treatment. Substantial effort has been expended toward exploiting various strategies to deliver theranostic agents to tumors, including passive and active targeting.1,2 In recent years, many other tumor-targeting approaches utilizing physical forces or stimuli, such as magnetic fields (MFs), light, and ultrasound, to enable the tumor-specific delivery of therapeutic agents have received significant interest because physical interactions are independent of the complicated cancer molecular biology pathways and the effects are more controllable and predictable. Iron oxide magnetic nanoparticles (MNPs) have excellent magnetic properties and good biocompatibility and have attracted extensive attention for their potential applications in tumor theranostic fields, such as T2-weighted magnetic resonance imaging (MRI),3-7 photothermal therapy,8 or therapeutic agent delivery.9 Tumor targeting using MNPs to carry therapeutic agents under external MF guidance has emerged as a promising tumor treatment approach because it does not require the specific binding of receptors expressed on tumor cells. Therefore, this approach could be applicable to a wide range of solid tumors.10 The MF-guided, targeted delivery of a cancer treatment was first reported in the 1970s and used magnetic albumin microspheres loaded with doxorubicin (DOX) in the 1970s.11 Since then, several improved MNP nanosystems have been developed and applied as novel delivery mechanisms for biomolecules (e.g., small molecules, proteins or genes) to treat cancer and other diseases.12-16 Despite very promising results in preclinical investigations, some clinical trials have shown that such systems have poor efficiency, and no magnetic nanocarriers have yet been approved.17,18 In recent years, most studies of magnet-related drug delivery have been based on small superparamagnetic iron oxide nanoparticles (SPIONs), which are often < 20 nm in diameter.19 Unfortunately, a real external magnetic field enhanced tumor targeting applications might not work as well as expected in vivo since there are other factors that must be considered in designing an effective MNP-based delivery system.20－22 The success of using MNPs for MF-guided tumor-targeted delivery depends on several aspects, such as having the optimal external MF density, a sufficiently long nanoparticle circulation time after systemic administration and nanoparticles with a strong magnetic responsiveness to the external MF during circulation.

21,22

These features are largely related to the surface physical 3

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properties and size of the chosen nanoparticles; size plays a very important role here. However, few systematic studies have been performed to evaluate the effect of MNP size on their application in MF-mediated, targeted delivery in vitro and in vivo. Thus, it is necessary to identify the optimal size range for nanoparticles to exert their biofunctions. In our previous study,23 we synthesized a series of monodisperse Fe3O4 nanoparticles ranging from 60 to 310 nm in size, and we systematically investigated the effect of their size on various biofunctions. The results indicated that nanoparticles 60 nm in diameter exhibited greater cellular internalization, while those120 nm in diameter were optimal for MRI and photoacoustic tomography in vitro. There were no significant differences among nanoparticles in the 60~310 nm size range in photothermal conversion efficiency when they were used as a photothermal agent. Herein, the relationship between Fe3O4 nanoparticle diameter across a wider range and MF-guided targeting efficiency was further investigated in vitro and in vivo. We focused on diameters of 60, 120, and 200 nm, which cover the typical size range, as well as diameters of 10 and 310 nm, which are close to the cutoff sizes for nanomaterial renal clearance (10 nm) and extravasation from the tumor vasculature (310 nm), respectively. Furthermore, the antitumor efficacy of the nanoparticles was assessed after they were loaded with DOX and irradiated with a near infrared (NIR) laser. In this way, we have aimed to provide a general idea of iron oxide nanoparticles sizes suitable for magnetic-targeting applications.

RESULTS Characterization of Fe3O4 nanoparticles Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 and Fe3O4-310 nanoparticles with similar chemical components and surface properties were prepared using a modified solvothermal reaction. The detailed parameters involved in the synthesis of the nanoparticles are listed in Table S1. All nanoparticles had a spherical shape, as viewed by transmission electron microscopy (TEM) (Figure S1A), and the nanoparticles were monodisperse with average sizes of 9.5 ± 1.3 nm, 54 ± 9.3 nm, 104 ± 10.6 nm, 186 ± 19.3 nm, and 296 ± 22.8 nm, respectively. The diameters determined by dynamic light scattering (DLS) was slightly larger than those determined by TEM; the average hydrodynamic sizes of the nanoparticles were 11.6 ± 1.1 nm, 59 ± 6.4 nm, 121 ± 4.8 nm, 198 ± 8.9 nm, and 309 ± 10.5 nm, respectively (Figure S1B). The zeta potential of the five types of 4 ACS Paragon Plus Environment

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nanoparticles ranged from -28.4 to -23.1 eV (Table 1). Representative diffractograms obtained by X-ray diffraction (XRD) are shown in Figure S1C (standard Fe3O4 and Fe3O4-10). The position and relative intensity of the characteristic diffraction peaks of Fe3O4-10 completely matched those of standard Fe3O4, indicating that nanoparticles were successfully synthesized. The XRD results of the Fe3O4 nanoparticles were consistent with those of the other four types of the Fe3O4 nanoparticles, as previously described.23 The representative magnetization curves of Fe3O4-10 nanoparticles were measured at room temperature, and the saturation magnetization value was 75.2 emu/g (Figure S1D), which is close to that of Fe3O4-120, as previously reported.23 Furthermore, the five types of nanoparticles dispersed in water and were stable for more than three months without any aggregation. Carboxymethyl chitosan (CMCTS)-coated Fe3O4 nanoparticles were formed through the reaction of carboxyl groups on the magnetic particles with amino groups on CMCTS. In our previous study, we determined that the Fe3O4 nnaoparticles could be successfully coated with CMCTS.24 DOX molecules were easily loaded onto the CMCTS-coated Fe3O4 nanoparticles via an electrostatic attraction by adjusting the solution pH from weakly acidic to either neutral or weakly alkaline conditions. Then, the DOX-loaded Fe3O4 nanoparticles were purified by being washed with phosphate-buffered saline (PBS, pH 7.4) until the supernatant became colorless. The drug-loading efficiency was calculated using the following formula: (the weight of loaded DOX / the weight of DOX-loaded nanoparticles) × 100%; for the five types of nanoparticles, this value ranged from 11.8 ~ 12.3%. Similar drug-loading efficiencies could be obtained for these five types of nanoparticles by adjusting the amount of DOX added when preparing the DOX-loaded nanoparticles. The DOX concentration ranged from 175 to 225 µg/mL. Fluorescent 6-courmarin or indocyanine green (ICG)-labeled Fe3O4 nanoparticles were also prepared using the methods described above. The storage stability of the DOX-loaded MNPs was examined over various time periods. We found that less than 10% of the loaded DOX molecules were released from the nanoparticles within 10 h when the nanoparticles were suspended in PBS (pH 7.4) at 37°C. However, only approximately 65% of the DOX remained intact after 24 h of incubation (Figure S2A). We further investigated the stability of the DOX-loaded MNPs in murine plasma at 37°C to simulate normal physiological blood conditions. As shown in Figure S2B, more than 90% of the loaded DOX remained after 6 h of incubation, indicating the stability of the system in plasma. In addition, there 5 ACS Paragon Plus Environment


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was no obvious change in the average hydrodynamic diameter of the nanoparticles in PBS or plasma measured by DLS for at least 1 day (Table S2 and Table S3). Figures S2C and S2D show the release profiles of DOX under different pH conditions in vitro. All the nanoparticles showed sustained and pH-dependent DOX release patterns. At pH 7.4, only 50% of the DOX molecules were released from DOX-loaded nanoparticles within 24 h, while more than 75% of the DOX molecules were released from the nanoparticles at pH 5.5. This result was probably due to the strong electrostatic attraction between DOX and the nanoparticles at physiological conditions. Furthermore, in an acidic environment, DOX was released significantly faster from the nanoparticles. The accelerated release profile under acidic conditions may be explained by the carboxyl groups on the CMCTS-coated MNPs, weakening the electrostatic coupling between DOX and CMCTS, and thus the Fe3O4 nanoparticles. Table 1. Characterization of Different-sized Fe3O4 Nanoparticles Dynamic Light Scattering Sample

Size (nm) (n=3)

Polydispersity index (PDI) (n=3)

Size (TEM) (nm) (n=100)

ζ-potential (eV) (n=3)

Fe3O4 - 10

11.6± 1.1

0.043 ± 0.027

9.5 ± 1.3

-28.4 ± 2.2

Fe3O4 - 60

59 ± 6.4

0.083 ± 0.037

54 ± 9.3

-27.4 ± 1.2

Fe3O4-120

121 ± 4.8

0.078 ± 0.003

104 ± 10.6

-26.1 ± 3.8

Fe3O4-200

198 ± 8.9

0.082 ± 0.043

186 ± 19.3

-23.1 ± 1.7

Fe3O4-310

309 ± 10.5

0.132 ± 0.048

296 ± 22.8

-26.8 ± 2.8

Magnetic responsiveness of Fe3O4 nanoparticles The different Fe3O4 nanoparticles were well dispersed in PBS (named Solution a) and a solution with a viscosity similar to that of blood (named Solution b), and the solutions become homogeneously brown before magnetic separation (Figure 1A). With increasing time under an external MF, the Fe3O4-60, Fe3O4-120, Fe3O4-200, and Fe3O4-310 solutions became increasingly colorless as the nanoparticles were collected near the magnet within a period of 5 min. However, there was only a slight change in the Fe3O4-10 solution under the same external MF for 48 h (Figures S3 and S4). These results indicated that the larger Fe3O4 nanoparticles move faster under the fuidance of the MF, exhibiting a higher magnetic responsiveness. For all the nanoparticles, the movement rate was significantly slower in Solution b due to the increased solution viscosity, 6 ACS Paragon Plus Environment

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indicating decreased nanoparticle magnetic responsiveness. The different DOX-loaded Fe3O4 nanoparticles exhibited a similar tends in responding to the external MF. The strong magnetic responsiveness of the DOX-loaded Fe3O4 nanoparticles with a diameter of 60~310 nm induced the rapid movement of DOX towards the magnet (Figures 1A, S5 and S6), which was further confirmed using a Maestro imaging system. DOX fluorescence was mainly present at the sidewall of the vial near the magnet after 12 h. However, because of the weak magnetic responsiveness of Fe3O4-10 nanoparticles, the same MF did not significantly change the DOX distribution in the solutions during the same time period (Figure 1B). To evaluate the magnetic-targeting ability of the Fe3O4 nanoparticles of different sizes, a microfluidic system that could control the nanoparticle solution flow rate was used to simulate nanoparticle retention in the blood circulation via the guidance of an external MF (Figure 1C). After circulating for 2 h, Fe3O4 nanoparticles were retained at the area of the tube wall where the magnet was attached the magnet. Compared with Solution a, Fe3O4 nanoparticles retention in Solution b significantly decreased for each size due to the increase in the viscosity (Figure 1D). Although the magnetic retention of the nanoparticles under flow was decreased in Solution b due to the high viscosity (Figures 1E, S7 and S8), the retention trend was consistent with that of nanoparticles in PBS; Fe3O4-310 exhibited the highest magnetic responsiveness. The retention tends of the five sizes of DOX-loaded Fe3O4 nanoparticles under flow in Solution a and b were similar to those of Fe3O4 nanoparticles (Figures S9 and S10). DOX exhibited with the highest magnet-guided accumulation under flow when loaded onto Fe3O4-310 nanoparticles (Figure 1F).

Cytotoxicity The

cytotoxicity

of

the

different

Fe3O4

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

nanoparticles bromide

was

(MTT)

determined method.

The

by

the

results

demonstrated that exposure to the nanoparticles at concentrations ranging from 0 to 2000 µg/mL for 24 h or 48 h induced very little MCF-7 cell death (Figure S11). The results also demonstrated that cell viability was slightly lower after the cells were exposed to an external MF, which can be attributed to the increased cellular uptake of nanoparticles with the external MF exposure. These results suggested the high biocompatibility of the five types of Fe3O4 nanoparticles. 7 ACS Paragon Plus Environment


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Figure 1. Magnetic responsiveness of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 and Fe3O4-310 nanoparticles in vitro. (A) Images of the different nanoparticles and DOX-loaded nanoparticles (in Solution a or Solution b) at equivalent Fe3O4 concentrations under an external MF over time. (B) Fluorescent images of the different DOX-loaded nanoparticles before and after magnetic separation. (C) Schematic of simulated blood circulation used for magnetic separation in vitro. (D) Magnetic retention of the different nanoparticles in the simulated blood circulation system over time (flow velocity: artery, 32.85 cm/s, in Solution a or Solution b). (E) The Fe mass of DOX-loaded nanoparticles in the tube near the magnet site after magnetic separation in (D). (F) The DOX mass of DOX-loaded nanoparticles in the tube near the magnet site after magnetic separation in (D). The data were analyzed by one-way analysis of variance (ANOVA) (*P ≤ 0.05; **P ≤ 0.01)

Cellular uptake of Fe3O4 nanoparticles The design of the MF that assisted the cellular uptake of the 6-coumarin-labeled, DOX-loaded Fe3O4 nanoparticles is shown in Figure 2A. MCF-7 cells were incubated with the nanoparticles for 0~48 h (Figure S12). The intracellular fluorescence of both 6-coumarin (Figure 2B) and DOX (Figure 2D) in the nanoparticles of each size was gradually increased with increased incubation time (0~24 h) and then was decreased at 48 h. The fluorescence was always well co-localized during the entire experiment (Figure S12). The cellular internalization of the nanoparticles increased and subsequently decreased during the incubation time, and DOX remained stably attached to the Fe3O4 nanoparticles within the cells for at least 48 h. Obviously, the external MF caused a significant increase in the cellular internalization of the nanoparticles of each size (Figures 2C and 2E). After 24 h of incubation under an MF, the average 6-coumarin florescence intensities in cells with the Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 and Fe3O4-310 nanoparticles were 1.05-, 1.38-, 1.62-, 1.74-, and 3.05-fold higher than those observed without a MF, respectively (Figures 2F and 2G). Although Fe3O4-310 exhibited the weakest cellular uptake, they exhibited the most rapid increase in cellular internalization in the presence of an MF because they had the strongest magnetic responsiveness. The Fe3O4 nanoparticles could assemble at the magnet site, thus delivering DOX to the same site (Figures 2C and 2E). The results indicated that the cellular uptake of DOX after 24 h of incubation with a MF was 1.13-, 1.35-, 1.53-, 1.68-, and 2.68-fold higher than that observed without an MF, 9 ACS Paragon Plus Environment


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respectively (Figures 2D, 2E, and 2H). Therefore, these MNPs could potentially be used for MF-enhanced targeting and drug delivery applications.

Figure 2. Magnet-assisted cellular internalization of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 and Fe3O4-310 nanoparticles by MCF-7 cells. (A) Schematic of the experimental process. (B) 6-Coumarin fluorescence intensity of cells after incubation with different nanoparticles at 37°C without a MF. (C) 6-Coumarin fluorescence intensity of cells after incubation with different nanoparticles at 37°C with an MF. (D) DOX fluorescence intensity of cells after incubation with different nanoparticles at 37°C without an MF. (E) DOX fluorescence intensity of cells after incubation with different nanoparticles at 37°C with an MF. (F) Confocal laser scanning microscopy images of MCF-7 cells after incubation with different nanoparticles (green) for 24 h with or without an external magnet under the cell culture dishes. Nuclei were stained with Hoechst 33258 (blue). 10 ACS Paragon Plus Environment

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Scale bar, 100 µm. (G) Semi-quantitative analysis of 6-coumarin fluorescence intensity in (F). (H) Semi-quantitative analysis of DOX fluorescence intensity in (F). All data are represented as the average ± SD; the data were analyzed by one-way ANOVA (*P ≤ 0.05; **P ≤ 0.01).

Biodistribution and magnet-guided tumor targeting We studied the MF-mediated tumor-targeting ability of the different Fe3O4 nanoparticles using an

in vivo bilateral tumor model with the left tumor under the influence of an external magnet (Figure 3A). Murine auto-fluorescence was removed by spectral unmixing using Maestro software. Over time, all fluorescence signals increased in the tumors, and the fluorescence intensities were maintained for less than 2 days (Figure 3B). Larger Fe3O4 nanoparticles showed greater accumulation in the tumors than did smaller nanoparticles, which was in contrast with the trend observed for the cellular uptake of the different Fe3O4 nanoparticles in vitro. As a control, mice injected with free ICG were also imaged. The results revealed no significant tumor uptake of ICG regardless of the presence of a MF. Furthermore, the bilateral tumors did not show obvious differences in nanoparticle accumulation without an MF (Figure 3C). Excitingly, the Fe3O4 nanoparticles (except for Fe3O4-10) tended to be enriched in the tumors under a MF (Figures S13 and 3C), in which the ICG fluorescence signals showed a significant increase over time after injection (Figures 3D and 3E). The biodistribution of Fe3O4-10 in the left tumors (+MF) and right tumors (-MF) were 4.0 ID%/g and 3.5 ID%/g at 48 h post-injection, respectively, and the difference between the number of nanoparticles in left and right tumors increased with increasing nanoparticles size. For Fe3O4-310, the tumor accumulation was 29.5 and 15.3 ID%/g on the left and right side, respectively. The MF induced 0.94, 1.26, 1.46, 1.82, and 1.94-fold increases in nanoparticle accumulation in tumors at 48 h after the injection of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200, and Fe3O4-310 nanoparticles, respectively, as determined by measuring the Fe content of in tumors using inductively coupled plasma–mass spectrometry (ICP-MS) (Figures 3F and 3G). Larger Fe3O4 nanoparticles clearly exhibited more efficient MF-enhanced tumor targeting. Our data indicated that 310 nm might be the optimal Fe3O4 nanoparticle diameter for retention in tumors targeted under MF guidance due to the strong magnetic responsiveness observed in vivo. The MF also mediated an increased accumulation of DOX in tumors when DOX loaded Fe3O4 nanoparticles were administered. At 48 h after the injection of DOX-loaded nanoparticles, strong 11 ACS Paragon Plus Environment


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DOX fluorescence was observed in tumors under the influence of an MF (left side), whereas weak DOX fluorescence was observed in tumors not under the influence of a MF (right side) (Figures 4A and S14). The semi-quantified intensity of DOX fluorescence observed in the confocal images indicated that the larger nanoparticles could deliver more DOX to tumors under the influence of an MF (Figures 4B and 4C). We further determined the DOX concentration within the tumors by extracting DOX from tumor lysates. Compared with free DOX, the DOX-loaded Fe3O4-10 nanoparticles did not significantly increase DOX accumulation in the tumors, even with MF guidance. The tumor DOX concentration was significantly (2-fold) higher after the injection of DOX-loaded Fe3O4-310 nanoparticles that after that of free DOX at the same dose. Importantly, DOX tumor accumulation further increased under external MF guidance and was 2.3-fold higher than that in the tumors without MF guidance and 5.1-fold higher than that after the administration of free DOX (Figure 4D). We further found that Fe3O4-310 nanoparticles might be a good carrier for targeted drug delivery with MF guidance.


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Figure 3. MF-enhanced biodistribution in vivo. (A) Experimental schematic of MF-enhanced biodistribution in vivo. The mice bearing bilateral tumors were divided into two groups, and the surfaces of tumors on the left side of mice in the “with MF” group were fitted with a magnet. (B) Fluorescence imaging of mice after the intravenous injection of the different Fe3O4 nanoparticles. Red circles indicate tumors fitted with a magnet. (C) Ex vivo imaging of tumors harvested from the mice at 48 h post-injection. (D) Quantification of the ex vivo fluorescence signals of tumors in the “without MF” group. (E) Quantification of the ex vivo fluorescence signals of tumors in the “with MF” group. (F) ICP-MS quantification of Fe in tumors in the “without MF” group. (G) ICP-MS quantification of Fe in tumors in “with MF” group. 13 ACS Paragon Plus Environment


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MRI for Fe3O4 nanoparticles The MRI contrast enhancement efficiencies of the different Fe3O4 nanoparticles were investigated using a clinical 3T MRI scanner. T1- and T2-weighted images of the nanoparticle suspensions are shown in Figures S15A and S15B. The T2 MR signal decreased with increasing nanoparticle concentration, suggesting that the nanoparticles enhance the transverse proton relaxation process. The R1 and R2 values (longitudinal and transverse relaxation rates; Table S4) varied linearly with the Fe concentration (Figures S15C and S15D). Furthermore, T2-weighted images of cells after incubation with the nanoparticles are shown in Figure S15E. Cellular internalization of the nanoparticles induced obvious changes in the T2-weighted MR images and T2 values (Figure S15F). Our data indicated that all five types of Fe3O4 nanoparticles are good candidates for T2 MRI contrast agents. The MF-mediated, tumor-targeted delivery of the different Fe3O4 nanoparticles was further investigated in a bilateral tumor model using MRI, which was conducted at different time points following nanoparticle injection. Fe3O4 nanoparticles tended to be enriched in the tumors fitted with a magnet (Figure 4E, bottom, green arrows); the T2 signals in these tumors decreased over time after injection (Figures 4E and S16). For all the nanoparticle sizes, the T2-weighted image intensity, calculated with GE Functool software, was remarkably lower in the tumors with a magnet than in the tumors without a magnet. In addition, larger Fe3O4 nanoparticles exhibited a greater difference in T2-weighted image intensity between the bilateral tumors (Figures 4G, S17B, S17D and 4I). Fe3O4-310 nanoparticles induced the lowest T2-weighted intensity in tumors under MF guidance; the intensity was 4.12-fold lower than that in tumors without MF guidance at 3 h post-injection. This observation was mainly attributed to the high nanoparticle accumulation with in the tumors and the high nanoparticle magnetic responsiveness (Figure 4I). In the controls, no significant differences in the T2-weighted intensity were found between the bilateral tumors under either MF condition (Figures 4F, S17A, S17C and 4H).


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Figure 4. MF-enhanced drug delivery to tumors and the MRI determination of magnetic targeting of cancer in vivo. (A) Confocal images of tumor sections prepared from the bilateral tumors of mice injected with Fe3O4 nanoparticles of different sized with or without MF guidance. Scale bar, 50 µm. (B) Semi-quantitative DOX fluorescence intensity of tumors in the “without MF” group in (A). (C) Semi-quantitative DOX fluorescence intensity of tumors in the “with MF” group in (A). (D) The DOX mass in the tumors (with or without MF) after injection of Fe3O4-10 or Fe3O4-310 nanoparticles. (E) In vivo T2-weighted MR images of mice after the intravenous injection of the different Fe3O4 15 ACS Paragon Plus Environment


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nanoparticles. Green and white arrows indicate tumors on the left and right sides, respectively. The left tumors in the lower panel were fitted with a magnet during the experiment. (F) T2-weighted MR image intensity of tumors without a magnet at 0 h. (G) T2-weighted MR image intensity of tumors with a magnet at 0 h. (H) T2-weighted MR image intensity of tumors without a magnet at 3 h post injection. (I) T2-weighted MR image intensity of tumors with a magnet at 3 h post-injection. All data are represented as the average ± SD; the data were analyzed by one-way ANOVA (*P ≤ 0.05; **P ≤ 0.01).

Figure 5. Photothermal effect and MF enhanced antitumor efficacy of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200, and Fe3O4-310 nanoparticles in vivo. (A) Thermographs of tumor-bearing mice that received photothermal treatment (808 nm, 1.5 W/cm2, 3 min) for different periods of time. (B) The images of tumors in the different groups at 30 days after treatment. (C) The growth curves of tumors were monitored over the course of the study (n = 6). (D) The body weight of mice was monitored over the course of the study. (E) The tumor weight in the control, Fe3O4-10, Fe3O4-10 plus MF, Fe3O4-310, and Fe3O4-310 plus MF groups after treatment. (F) The tumor weight in the control, free DOX, DOX-loaded Fe3O4-10, DOX-loaded Fe3O4-10 plus MF, DOX-loaded Fe3O4-310, and DOX-loaded Fe3O4-310 plus MF groups after treatment. (G) Hematoxylin and eosin (H&E) stained sections of tumors from different groups after treatment. Scale bar, 50 µm. All data were analyzed by one-way ANOVA (*P ≤ 0.05; **P ≤ 0.01). 16 ACS Paragon Plus Environment

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Photothermal conversion in vivo With the injection of the different Fe3O4 nanoparticles into mice bearing S180 tumors and NIR laser irradiation, the tumor temperature quickly rose and then plateaued; however, with the injection of saline, the tumor temperature only slightly increased under the same irradiation conditions (Figure 5A). The average tumor temperature was measured by analyzing infrared thermal images using FLIR QuickReport software. Fe3O4-310 generated the highest tumor temperature (57.3°C) under NIR laser irradiation because these nanoparticles exhibited the highest tumor accumulation.

In vivo anticancer efficacy study MF-mediated photothermal and chemotherapy were performed against S180 tumors in ICR mice after the injection of DOX-loaded Fe3O4 nanoparticles, and all tumors were subsequently irradiated with an NIR laser (1.5 W/cm2 for 3 min, 3 irradiations). Images and growth curves of the tumors after treatment are shown in Figures 5B and 5C. Tumors in the control group (treated with saline) grew rapidly, and the tumor volume was approximately 10-fold higher on day 30 than on day 0. The MF did not enhance the tumor retention or therapeutic effect of the Fe3O4-10 nanoparticle, inducing only slight tumor growth inhibition (TGI). However, because the MF could obviously increase the tumor accumulation of Fe3O4-310 nanoparticles, tumors in the Fe3O4-310 plus MF group were much smaller than those in the Fe3O4-310 group. The external MF more efficiently enhanced the antitumor efficacy of Fe3O4-310 than Fe3O4-10, which could be mainly be attributed to the higher in vivo magnetic responsiveness of Fe3O4-310. Furthermore, Fe3O4-310 could deliver high levels of DOX into the tumors in the presence of an external MF. Thus, the best TGI was observed in the DOX-loaded Fe3O4-310 plus MF group. The tumor weights in all the groups at the end of the experiment are shown in Figures 5E and 5F. Tumor growth was strongly inhibited in the DOX-loaded Fe3O4-310 and DOX-loaded Fe3O4-310 plus MF groups, and the tumors had nearly disappeared by day 30. We also investigated the therapeutic effect of Fe3O4-200 nanoparticles in the presence of an MF. Predictably, due to the higher magnetic responsiveness, the magnetic targeting and TGI of Fe3O4-200 were superior to those of Fe3O4-10 (Figure S18). The body weight of the mice dropped slightly from day 0 to day 3 in several groups and then consistently increased thereafter (Figure 5D), suggesting low systemic toxicity for all treatments. 17 ACS Paragon Plus Environment


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Sections prepared from the of major organs (heart, liver, spleen, lungs, and kidneys) of mice in each treatment group (except for the free DOX group) showed no noticeable abnormalities or lesions compared with those from mice in the saline group, indicating a lack of appreciable organ damage and further suggesting the limited toxicity of the nanoparticles (Figure S19). The microvessel density of the residual tumor tissue in each group was assayed at the end of the experiment. Significantly fewer positive brown cells, indicating vessel epithelial cells, were observed in the Fe3O4-310 treatment groups (i.e., the Fe3O4-310, DOX-loaded Fe3O4-310, Fe3O4-310 plus MF, and DOX-loaded Fe3O4-310 plus MF groups) than in the other groups (Figures 6A, 6B, and 6C). Ki67 staining also showed that the lowest tumor cell proliferation occurred in the Fe3O4-310 treatment groups (Figure 6D), and this result was confirmed by calculating the proliferation index of the tumor sections (Figures 6E and 6F). More necrotic and apoptotic cells were found on the tumor sections of these groups (Figures 6G, 6H and 6I). These data demonstrated that Fe3O4-310 exhibited a significantly enhanced therapeutic effect under the guidance of an external MF. These results clearly indicated that the larger Fe3O4 nanoparticles had a greater antitumor efficacy than did the smaller Fe3O4 nanoparticles in the magnet-assisted treatment.


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Figure 6. Histological analysis of tumor tissues after the long-term efficacy study of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200, and Fe3O4-310 nanoparticles in vivo. (A) S180 tumors were stained with CD31 to analyze tumor angiogenesis. Representative images of CD31 staining are shown. Scale bar, 50 µm. (B and C) Quantification of microvessel density staining in different groups after treatment. (D) S180 tumors were stained with Ki67 to analyze tumor cell proliferation. Representative images of Ki67 staining are shown. Scale bar, 50 µm. (E and F) Quantification of Ki67 staining in different groups after treatment. (G) S180 tumors were stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to analyze tumor cell proliferation. Representative images of TUNEL staining are shown. Scale bar, 50 µm. (H and I) Quantification of TUNEL staining in different groups after treatment. All data were analyzed by one-way ANOVA (*P ≤ 0.05; **P ≤ 0.01).



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DISCUSSION The unique physical properties of MNPs enable them to serve simultaneously as imaging probes for locating and diagnosing cancerous lesions and, as drug vehicles for preferentially delivering therapeutic agents to those lesions. MNPs with a proper diameter can efficiently and passively accumulate in many tumor tissues via the enhanced permeability and retention effect. Importantly, unlike other types of conventional nanoparticles (e.g., liposomes, polymeric micelles, or gold nanoparticles), MNPs can easily obtain enhanced targeting delivery to the lesion location, such as tumors, via mediation of an external MF almost without any invasion based on their responsiveness to the MF. Therefore, MNPs loaded with a drug of interest can also be guided to a specific tissue or organ by applying an external MF, thereby achieving a high drug concentration in a diseased area.25 Although many studies have reported the application of MF-mediated targeting based on MNPs, the barrier to its wide application has not been addressed. The MF-mediated targeting effect depends on the magnetic responsiveness of MNPs and is further related to the attractive force (FMNP) of an MF to the nanoparticles, which can be calculated according to the following formula:

‫ܨ‬ெே௉ = (∇‫ܤ‬ଶ )

χܸ௖ 2μ଴

where ∇B is the gradient produced by an externally applied MF (B) to a tumor region, χ is the magnetic susceptibility of the magnetic nanoparticle, Vc is the volume of the core, and µ0 is the magnetic permeability of free space. MNPs can be retained in a tumor site when the magnetic force applied to the tumors is sufficient to overcome the hydrodynamic drag forces exerted on the nanoparticles by blood flow.26 Therefore, according to the formula, the magnetic responsiveness is directly related to the nanoparticle size. Most magnetic nanosystems have been constructed based on very small (< 20 nm) SPIONs, and it might be difficult to capture these particles with an MF due to their low magnetic responsiveness.20-22 However, smaller particles may exhibit faster clearance in vivo and thus have a limited circulation time for interacting with the applied field, thereby inducing decreased MF-guided, targeted delivery.27,28 Although the optimal size range of MNPs for magnetic targeting needs to be investigated, studies on the effect of MNP size on their bio-applications have examined relatively small size ranges. In this study, Fe3O4 nanoparticles with a much broader size range (10-310 nm) were synthesized. To examine whether the MNPs could carry drug molecules to target sites, DOX-loaded Fe3O4 20 ACS Paragon Plus Environment

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nanoparticles were also prepared. First, we investigated the magnetic responsiveness of Fe3O4 nanoparticles of different sizes in a static solution system and a simulated blood circulation system, where PBS and a solution with a viscosity similar to that of blood were chosen as the media and denoted as Solution a and b, respectively (Figure 1). Our data indicated that the Fe3O4 nanoparticles exhibited significantly decreased magnetic responsiveness when they were suspended in the solution with high a higher viscosity. Furthermore, the smaller nanoparticles were much more difficult to capture with the magnet in the static and dynamic systems. The larger nanoparticles not only exhibited an enhanced magnetic responsiveness, but the delivery of their payload (i.e., DOX) to the target site could be enhanced via MF guidance (Figures S3-S10). The data further demonstrated that the Fe3O4 nanoparticles 310 nm in diameter exhibited magnetic responsiveness superior to that of the smaller nanoparticles, suggesting that Fe3O4-310 or DOX-loaded Fe3O4-310 nanoparticles would have a better magnetic-targeting effect in vivo.29 To investigate the in vivo MF-guided, tumor-targeting ability of Fe3O4 nanoparticles, a dual tumor model was employed. The left tumors were fitted to a magnet, while the right tumors were used as the control and not fitted with a magnet (Figure 3A). The fluorescence signal, T2-weighted intensity and Fe mass in the tumor site after the injection of the different Fe3O4 nanoparticles were measured using an in vivo Maestro imaging system, MRI and ICP-MS (Figures 3 and 4). The results confirmed that the larger nanoparticles exhibited greater tumor accumulation and retention (Figures 3D and 3F). The tumor retention and accumulation could be further increased under the guidance of an external MF in the tumors (Figures 3E and 3G). Furthermore, the magnetic-targeting efficiency mediated via an external MF was greater for the larger Fe3O4 nanoparticles, and the DOX-loaded Fe3O4 nanoparticles exhibited tumor accumulation and magnetic-targeting trends similar to those of the nanoparticles not loaded with DOX (Figures 4A-4D). This result indicated that DOX could be loaded onto the nanoparticles and delivered to a tumor site via MF guidance. In this way, a high DOX concentration can be obtained in a region of interest under external MF mediation. Our data indicated that of the investigated sizes, 310 nm might be an optimal nanoparticle size for magnetic targeting applications due to the higher tumor accumulation and magnetic responsiveness observed. The MF-mediated therapeutic efficacy of DOX-loaded Fe3O4 nanoparticles depended on their magnetic responsiveness, accumulation level and tumor extravasation. For photothermal therapy, 21 ACS Paragon Plus Environment


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the high accumulation of photothermal conduction agents in tumors should be preferentially considered over the uptake of the agents by tumor cells. For magnetic-targeted delivery, nanoparticles should exhibit excellent magnetic responsiveness in vivo. Our data demonstrated that the in vivo photothermal ablation and chemotherapy efficacy under NIR laser irradiation after the intravenous injection of unloaded Fe3O4 nanoparticles or DOX-loaded Fe3O4 nanoparticles increased with increasing particle size over the range investigated here (Figures 5B, 5C, and S18). The DOX-loaded Fe3O4-310 plus MF group showed the strongest antitumor activity in response to NIR laser irradiation (Figure 6) because the Fe3O4-310 nanoparticles had the highest accumulation at the tumor site and the most sensitive magnetic response (Figures 3 and 4).

CONCLUSION We synthesized a series of monodisperse Fe3O4 nanoparticles with identical surface properties, ranging in size from 10 to 310 nm, and we systematically investigated their behavior and magnet-assisted antitumor efficacy. Our data indicated that smaller Fe3O4 nanoparticles exhibited greater cellular internalization, while larger Fe3O4 nanoparticles showed greater tumor accumulation. Stronger magnetic responsivenesswas exhibited by larger Fe3O4 nanoparticles in vitro and in vivo, and this responsiveness could induce increased nanoparticle tumor accumulation and payload (i.e., DOX) delivery to the tumor site under the guidance of an external MF. Our study demonstrated that Fe3O4-310 nanoparticles exhibited the best magnetic-targeting efficiency mediated by an external magnetic

field,

inducing

the

strongest

antitumor

efficacy

with

combination

photothermal-chemotherapy. Our results could serve as a valuable reference for the future design of MNPs and targeted delivery systems mediated by an external MF.

MATERIALS AND METHODS Reagents Iron (III) chloride hexahydrate (FeCl3•6H2O), trisodium citrate (Na3Cit), sodium acetate trihydrate (NaAC•3H2O), ethylene glycol, and ferrous sulfate (FeSO4•7H2O), were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Polyethylene glycol (Mw = 4000) and ICG were purchased from Aladdin Co., Ltd. (Shanghai, China). Sodium linoleate, linoleic acid, Hoechst 33258, MTT, and 6-coumarin were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 22 ACS Paragon Plus Environment

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1,10-Phenanthroline monohydrate and hydroxylammonium chloride were purchased from Shanghai Titan Chemical Co., Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX•HCl) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). RPMI 1640 medium (RPMI), fetal bovine serum (FBS), and penicillin/streptomycin (100 U/mL) were from Ji Nuo Biotechnology Co., Ltd. (Zhejiang, China). All other chemicals were of analytical grade and were used without further purification. The deionized water used in all experiments was prepared using a Milli-Q system (Millipore, Boston, USA).

Cell lines and animals MCF-7 (human breast cancer) and S180 (mouse osteosarcoma) cells were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (IBCB, Shanghai, China). The cells were maintained in RPMI 1640 medium containing 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Male BALB/c nude mice (4-5 weeks old, 18 ± 2 g) and ICR mice (4-5 weeks old, 18 ± 2 g) were utilized according to the protocol approved by the Institutional Animal Care and Use Committee of Zhejiang University. In vivo experiments were performed in compliance with the Zhejiang University Animal Study Committee's requirements for the care and use of laboratory animals in research.

Preparation and characterization of Fe3O4 nanoparticles To prepare Fe3O4 nanoparticles with a diameter of 10 nm (Fe3O4-10), iron (III) chloride (FeCl3•6H2O) (4.0 mol/L) and ferrous sulfate (FeSO4•7H2O) (2.0 mol/L) were first diluted in 40 mL of ethanol; then, 1.6 g of sodium linoleate and 8 mL of linoleic acid were added to the solution. The mixture was stirred and transferred to a 70-mL autoclave tube. The reactions were controlled at 160°C for 10 h. After the mixture was cooled to room temperature, the products were collected at the bottom of the vessel.30 The other four types of Fe3O4 nanoparticles with diameters of 60 nm (Fe3O4-60), 120 nm (Fe3O4-120), 200 nm (Fe3O4-200), and 310 nm (Fe3O4-310), were synthesized using a previously reported solvothermal reaction.23,31 The detailed parameters involved in the synthesis of the nanoparticles are listed in Table S1. The resulting precipitates were washed several times with ethanol and deionized water. To increase the stability of the Fe3O4 nanoparticles in aqueous media, the nanoparticles were modified 23 ACS Paragon Plus Environment


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with carboxymethyl chitosan (CMCTS). Briefly, Fe3O4 nanoparticles (100 mg) and 12 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were dispersed in 5 mL of PBS (pH 6.0), followed by sonication for 20 min. Then, the mixture was sonicated for another 60 min after 10 mL of a CMCTS solution (20 mg/mL) was added. CMCTS-modified Fe3O4 nanoparticles were collected using a magnet and purified by several washes with PBS. The shape and size of the nanoparticles was determined by TEM (JEM-1230, Japan) and DLS (Malvern Nano-ZS 90, Malvern, UK). The XRD pattern of the Fe3O4-10 nanoparticles was recorded using an X’Pert Pro (PANalytical, The Netherlands) diffraction meter. The magnetic properties were measured using a vibrating sample magnetometer (MPMS-XL-5, Quantum Design, USA). DOX was loaded onto Fe3O4 nanoparticles by mixing CMCTS coated Fe3O4 nanoparticles (400 µg/mL, 10 mL) with a DOX solution (200 µg/mL, 10 mL) at room temperature for 12 h. The final product was washed three times with PBS for purification. 6-Coumarin- or ICG-labeled Fe3O4 nanoparticles were also prepared according to a previously reported method.23 The storage stability of the different DOX-loaded Fe3O4 nanoparticles was assessed as follows: freshly prepared DOX-loaded MNPs were stored for 10 and 24 h at 37°C, and then the MNPs were removed by centrifugation. Subsequently, the DOX present in the supernatant was determined by UV-Vis spectroscopy at 480 nm. To investigate the stability of the nanoparticles under physiological conditions, a DOX-loaded MNP solution was added to murine plasma at a final DOX concentration of 100 µg/mL, and the samples were incubated at 37°C. Aliquots were removed at 0, 1, 3, 6, and 24 h; MNPs were removed by centrifugation and then analyzed using UV-Vis spectroscopy. DOX retention was calculated using the weight ratio between the drug retained within the nanoparticles and that added at 0 h. The size distribution of the DOX-loaded nanoparticles dispersed in PBS and plasma from was also determined. The behavior of DOX release from nanoparticles was investigated at 37°C under different pH conditions. DOX-loaded nanoparticle solutions (1 mL, 7 mg/mL) were incubated in 5 mL of PBS (pH 7.4 and 5.5) at 37°C under gentle shaking (100 rpm). At predetermined time intervals, the supernatant was collected to determine the amount of drug released using UV-Vis. The medium was completely removed and then replaces with fresh PBS. All drug release tests were performed three times. 24 ACS Paragon Plus Environment

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Magnetic responsiveness of Fe3O4 nanoparticles A solution with a viscosity similar to that of blood (denoted Solution b) was prepared by adding CMCTS to a PBS solution (viscosity: 6.0522×10-3 Pa•s). The nanoparticles were diluted with Solution a (PBS) or Solution b to 7 mL (at a concentration of 300 µg/mL) and then added to vials. The vials were then placed next to a magnet (MF density: 0.5 T) for selected time intervals. The separation processes were recorded using a camera. For the same experiment was performed using DOX-loaded nanoparticles, and the DOX fluorescence in the vials was monitored with a Maestro imaging system (CRI, Inc., Woburn, MA, USA). To evaluate the magnetic responsiveness of the nanoparticles in the in vivo environment, the blood circulation was simulated using a microfluidic system, as shown in Figure 1C. The different nanoparticles were added to the dynamic flow system in Solution a or Solution b, and the liquid flow rate was controlled and held at 32.85 cm/s, which is close to the velocity of arterial blood flow. A magnet (MF density: 0.5 T) was placed under the pipe. The process was recorded with a camera. After a few minutes in circulation, the nanoparticle retention by the magnet was observed, and the Fe ion level was quantified via a spectrophotometric method with 1,10-phenanthroline monohydrate. The samples were also treated with ultra sound for DOX extraction and the amount of DOX was determined by fluorescence spectrophotometry (Hitachi-7000, Japan).

Cytotoxicity The cytotoxicity of the different Fe3O4 nanoparticles was evaluated by MTT assays. MCF-7 cells were incubated with Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 or Fe3O4-310 nanoparticles at various concentrations with or without a magnet (MF density: 0.5 T). Cells cultured without Fe3O4 nanoparticles were used as a control. After 24 and 48 h, the Fe3O4 nanoparticles were removed, MTT solution was added, and cell viability was measured as the absorbance ratio of the test and control wells.

Cellular uptake Fe3O4 nanoparticles were labeled with 6-coumarin; then, DOX was loaded onto the nanoparticles. MCF-7 cells were incubated with the nanoparticles for different times. To investigate the impact of an external MF on the cellular uptake of the nanoparticles, a magnet (MF density: 0.5 T) was placed 25 ACS Paragon Plus Environment


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under the center of the cell culture dish. After incubation, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min and then observed by confocal microscopy (Zeiss, 710, LSM, Germany).

Biodistribution and MF guided targeting The animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of Zhejiang University. S180 cells were suspended in serum free RPMI 1640 medium and subcutaneously injected into the right rear flank of nude or ICR mice (5×106 cells per mouse). Tumors with a volume of 250-300 mm3 were used for the experiments. For real-time in vivo optical imaging of magnetic targeting, bilateral tumor-bearing nude mice were divided into two groups and intravenously injected with ICG-labeled Fe3O4 nanoparticles of different sizes (5 mg/mL, 200 µL per mouse). For mice in the magnetic-targeting group, a magnet (MF density: 0.5 T) was fitted to the surface of the left tumor using Steri-Strip tape, and the magnet was removed at 48 h after injection. As controls, mice in the other group were treated in the same manner, but no magnet was attached to the tumors. The whole-animal biodistribution of the nanoparticles was investigated in real time using a Maestro imaging system. Then, the mice were euthanized, and the tumors were harvested, washed with PBS, and placed in a dish. Fluorescence imaging was also performed using a Maestro imaging system. The Fe tumor content was determined by ICP-MS (Elan DRC II PerkinElmer, Waltham, MA, USA). After the tumors were weighed and then lyophilized, the samples were digested with 8 mL of aqua regia in 20-mL glass vials at boiling temperature. The solution was evaporated, and the precipitate was suspended in an aqueous solution containing 1.5% (v/v) HCl and 0.5% (v/v) HNO3. The suspension was centrifuged at 10,000 rpm for 5 min to remove any undigested debris. The Fe content in the supernatant was analyzed using ICP-MS. To investigate the MF-mediated accumulation of DOX in the tumor tissue, mice were treated with DOX-loaded Fe3O4 nanoparticles with or without a magnet fitted onto the surface of the tumors, as described above. After 48 h, the magnet was removed from the mice. The tumors were collected and embedded in optimal cutting temperature (OCT); then, they were sliced into 5-µm frozen slices for observation with by confocal microscopy. 26 ACS Paragon Plus Environment

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The DOX content in the tumors with or without external MF guidance was also determined. Briefly, bilateral S180 tumor-bearing nude mice were intravenously injected with free DOX, DOX-loaded Fe3O4-10 or DOX-loaded Fe3O4-310. Then, the left tumor was fitted with a magnet. After 48 h, the mice were euthanized and the tumors were harvested, washed with PBS, stored in liquid nitrogen, and triturated in a mortar. The powder was then dissolved in a borate buffer solution and treated with ultrasound. DOX was extracted by the addition of chloroform, and the DOX content was measured by fluorescence spectrophotometry.

MRI for Fe3O4 nanoparticles In vitro T1 and T2 measurements were acquired from a series of solutions containing cells and Fe3O4 nanoparticles concentrations ranging from 0~0.080 mM of Fe after incubation using multiple flip angle 3D SPGR and 2D multi-echo SE sequences using a 3T clinical MRI scanner (GE, discovery, USA). The MF-guided tumor targeting of Fe3O4 nanoparticles was further evaluated using MRI in vivo. Briefly, bilateral S180 tumor-bearing nude mice were intravenously injected with Fe3O4 nanoparticles of different sizes (5 mg/mL, 200 µL per mice). The left tumors (Figure 4E, bottom, green arrows) were fitted with a magnet (MF density: 0.5 T). The control mice were treated in the same manner but were not fitted with a magnet. Mice were imaged with a clinical MRI scanner at predetermined times after injection. A T2-weighted SE PROPELLER sequence was used for these in vivo scans, and the tumor region of interest was manually drawn on each T2-weighted image for further analysis of the temporal change in the image signal.

Photothermal conversion in vivo Nude mice bearing S180 tumors were injected with Fe3O4 nanoparticles of different sizes (5 mg/mL, 200 µL per mice) or the same volume of saline. After 24 h, the tumors were irradiated with an NIR laser (808 nm, 1.5 W/cm2, 3 min). The tumor temperature during irradiation was monitored using an infrared thermal imaging camera (FLIR ThermaCAM S65, USA). The resulting thermographs were analyzed using IR Flash software (Infrared Camera Inc., version 2.10) to obtain the average tumor temperature at each time point. 27 ACS Paragon Plus Environment


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Anticancer efficacy in vivo ICR mice bearing S180 tumors were randomly divided into ten groups (n = 6 per group) and treated with saline (control group), free DOX, Fe3O4-10, Fe3O4-10 plus MF, DOX-loaded Fe3O4-10, DOX-loaded Fe3O4-10 plus MF, Fe3O4-310, Fe3O4-310 plus MF, DOX-loaded Fe3O4-310, or DOX-loaded Fe3O4-310 plus MF (DOX dose: 5 mg/kg, nanoparticle dose: 200 µL per mice, 5 mg/mL; MF density: 0.5 T), besides, the dose of DOX or pure nanoparticles for all groups were the same. To systematically investigate the effect of nanoparticle size on magnetic targeting, we also investigated the magnet-assisted photothermal therapy efficiency of Fe3O4-120 nanoparticles. All tumors were irradiated with an NIR laser (808 nm, 1.5 W/cm2, 3 min) on the second day after injection. The tumor size was measured based on the width and length determined using a caliper. The tumor volume and animal body weight were measured every three days, and the tumor volume was calculated using the formula (length × width2 / 2). TGI (%) was calculated using the following formula: (1 - average tumor volume of treated group/ average tumor volume of control group) × 100%. Mice were sacrificed on day 30 after injection, and the tumors were collected, weighed, fixed in 4% paraformaldehyde, and cut into 5-µm sections for Ki67 and TUNEL staining to determine the proliferation and apoptosis indexes of the tumor cells. Ki67 or TUNEL positive cells were detected in randomly selected fields of the tumor sections. Both the Ki67 proliferation index and TUNEL apoptosis index were calculated by counting the number of brown (positive) cells among 1000 cells in field from 10 sections, and they were presented as the average ratio. The tumor slides were also stained for CD31 to examine the microvessel density. Other organs, including the heart, liver, spleen, lungs and kidneys, were fixed, stained with H&E and examined by light microscopy.

Statistical analysis The results are presented as the mean ± SD. Statistical comparisons were conducted using paired t-tests with a two-tailed p value to compare selected data pairs when only two groups were compared. If more than two groups were compared, an evaluation of significance was performed using a one-way ANOVA. All statistical analyses were conducted using GraphPad Prism software.

ASSOCIATED CONTENT 28 ACS Paragon Plus Environment

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Supporting information available Characterization of Fe3O4-10, Fe3O4-60, Fe3O4-120, Fe3O4-200 and Fe3O4-310 nanoparticles; characterization of DOX-loaded magnetic nanoparticles; photos of different sized nanoparticle (or DOX-loaded nanoparticles) solutions with the same concentration of Fe3O4 under the external magnetic field over time; magnetic retention of different sized nanoparticles (or DOX-loaded nanoparticles) in simulated blood circulation system over time; cytotoxicity of MCF-7 cells after incubation with nanoparticles; cellular uptake of MCF-7 cells incubated with different nanoparticles; fluorescence imaging of mice bearing bilateral subcutaneous S180 tumors on opposite flanks after intravenously injection of ICG labeled different sized Fe3O4 nanoparticles; magnetic resonance imaging study of different sized Fe3O4 nanoparticles; H&E staining of major organs sections obtained from mice models. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * To whom the correspondence should be addressed. Fax: +86 88208443. E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflict of Interest The authors declare no competing financial interest.

Acknowledgement This work was supported by the National Nature Science Foundation of China (81373348 and 81573365).

REFEENCES AND NOTES 29 ACS Paragon Plus Environment


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Curie

Temperature-Cancer

Theranostics

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TABLE OF CONTENTS GRAPHIC


External Magnetic Field-Enhanced Chemo-Photothermal Combination

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