Tunable Fe3O4 Nanorods for Enhanced Magnetic Hyperthermia Performance

Magnetic hyperthermia is one of the most promising techniques for treating gynecological cancer, where magnetite (Fe3O4) is the most common nanomaterial used as a magnetic hyperthermia agent. Here, we demonstrate that optimal Fe3O4 nanorods (NRs) can act as a magnetic hyperthermia agent with higher specific absorption rate (SAR), which is mostly attributed to their enhanced surface anisotropy. As a result, Fe3O4 NRs could effectively hinder the growth of gynecological cancer cells in nude mice models, again demonstrating its good magnetic heating properties. These results provide a powerful basis for the development of an ideal magnetic hyperthermia agent with enhanced SAR, thereby effectively treating gynecological cancer in clinical practice.

. First, 6.0 mM FeCl 3 · 6H 2 O was added to 60 mL of deionized water and stirring continued for 0.5 h to form a homogeneous solution, then transferred to a Teflon-lined stainless-steel autoclave and reacted at 100 °C for 4, 6, and 10 h. The β-FeOOH precursors were collected by centrifugation and washed with deionized water and alcohol three times, then dried at 60 °C. Second, 20 mg of different sized β-FeOOH NRs was uniformly dispersed in 6 mL trioctylamine, then 200 μL oleic acid was added to the mixture and kept stirring for 2 h, respectively. Then the yellow gelatinous mixture was collected by centrifugation at 7500 rpm for 10 min. Finally, under the flow of mixed gas of 95% Ar and 5% H 2 gas, the as-prepared precursors were transferred to a tube furnace for the reduction reaction at 340 °C and kept for 2 h, then naturally cooled to room temperature. The different sizes of Fe 3 O 4 NRs were collected though centrifuging, washed with hexane, and dried at 60 °C. The 460, 350, and 250 nm Fe 3 O 4 NRs correspond to different hydrothermal times of 4, 6, and 10 h, respectively. Synthesis of fe 3 o 4 nRs-Go. First, the as-prepared Fe 3 O 4 NRs was dissolved in chloroform at a concentration of 10 mg/mL, and 5 mg/mL aqueous solution of graphene oxide (GO) was prepared. Then octadecylamine (20 mg) and chloroform (1 mL) were fully dissolved by ultrasound for 1-2 min, then Fe 3 O 4 NRs solution (100 μL), deionized water (4 mL) and GO (1 mL) were added to further ultrasound for 30 min. Finally, the obtained solution was placed on a shaker for 6 h at 55 °C to remove chloroform, then the Fe 3 O 4 NRs-GO dispersion system was prepared.
Materials characterization. The phase structure, morphology, and chemical composition on the surface of the Fe 3 O 4 NRs were analyzed by X-ray diffraction (XRD) patterns (X'pert Pro Philips), scanning electron microscope (SEM, Hitachi S-4800), transmission electron microscope (TEM, Tecnai G2 F30), and X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra), respectively. Vibrating sample magnetometer (VSM Model EV9, MicroSense, LLC) and SQUID systems (MPMSXL-7) were carried out to measure hysteresis curves and zero field cooled (ZFC) and field cooled (FC) magnetization plots. Further, the hydrodynamic diameters and zeta potential of samples were measured on a Malvern Zetasizer Nano-ZS. The magnetic heating properties of Fe 3 O 4 NRs were analyzed by a radiofrequency heating machine (EASYHEAT-5060, Ambrell), the specific absorption rate (SAR) value was calculated based on the following equation: is the initial slope of the temperature versus time curve, C is the specific heat capacity of water, and m Fe is the weight fraction of Fe in solution.  www.nature.com/scientificreports www.nature.com/scientificreports/ In vivo experimental sequences. Sixteen female 6-8 weeks old nude mice, weighing about 20 g, purchased from Beijing Weitong lihua Experimental Animal Technology Co., Ltd, were kept in a sterile environment. Four mice were placed in each autoclaved cage and the animals were fed with a special diet, given water, and kept at an appropriate temperature. 0.1 mL of cancer cell suspension (about 1*107 cells) was injected subcutaneously into the right lower extremities of mice to induce tumors and observe the growth of mice. Then mice were randomly divided into 4 groups with a tumor nodule volume as long as about 80 mm 3 , and 4 mice in each group. Nude mice in the experimental group were injected subcutaneously with 0.1 mL (Fe concentration of about 1.2 mg/mL) of Fe 3 O 4 NRs-GO-AFM and Fe 3 O 4 NRs-GO, respectively. While in the control group, nude mice were injected with 0.1 mL of PBS buffer. Before treatment, the initial tumor volume of each nude mouse was measured and recorded (Volume = length * width * Width/2) 36,37 . Then three groups of nude mice were placed under a magnetic field of 308 Oe for 10 min. Finally, nude mice were sacrificed after 20 days, and tumor volume changes of each nude mouse were measured every 2 days before euthanasia.

Results and discussion
Different size of FeOOH NRs were prepared by a simple hydrothermal method with various reaction times. Figure 1a shows the X-ray diffraction (XRD) patterns of the prepared precursors of FeOOH NRs, all the diffraction peaks are well indexed to the β-FeOOH (JCPPS no. 75-1594). Figure 1b-d illustrate the scanning electron microscope (SEM) images of FeOOH NRs with lengths of 460 nm, 350 nm, and 250 nm, respectively, which correspond to the experimental conditions of 4 h, 6 h, and 10 h hydrothermal times, respectively.
After annealing at 340 °C for 2 h in a mixed gas of 95% Ar and 5% H 2 , FeOOH precursors were reduced to Fe 3 O 4 NRs as confirmed by XRD patterns (Fig. 2a). All the diffraction patterns of three samples correspond to the  Subsequently, a vibrating sample magnetometer (VSM) was used to estimate the magnetic properties of Fe 3 O 4 NRs. Figure 4a shows the room temperature M(H) hysteresis loop of samples, the saturation magnetization (M s ) of 460, 350, and 250 nm Fe 3 O 4 NRs are measured to be 72, 71 and 77 emu/g, respectively. Almost equal coercivity (H c ) and remanence (M r ) were obtained for three samples. However, the M s value is not changed significantly with the change of size for Fe 3 O 4 NRs, and lower than that of bulk Fe 3 O 4 (90 emu/g) at room temperature, which is possibly caused by the tiny uncompensated surface spin or the disordered surface microstructures 40 . The ZFC and FC magnetization plot of three Fe 3 O 4 NRs samples were measured to further confirm the phase structure and crystallinity of Fe 3 O 4 . The Verwey transition temperature (T v ) is detected at 120 K as shown in Fig. 4b, which is one of the criteria for judging Fe 3 O 4 41,42 , in accordance with the above XRD, XPS results. To further understand the hyperthermia performance of samples, the transformation of the aqueous phase was first performed to evenly disperse the Fe 3 O 4 NRs in water 43 . Then the magnetic properties of as-prepared samples were again analyzed as shown in Fig. 4a, the M s of 350 nm Fe 3 O 4 NRs-GO reaches a maximum of 60 emu/g, suggesting it has the best hyperthermia performance 44 . Besides, Fig. 4c shows that the Fe 3 O 4 NRs-GO dispersion is without obvious aggregation and stability, which is demonstrated by hydrodynamic diameter of 370 nm and zeta-potential of 50.1 mV, and the morphology of Fe 3 O 4 NRs-GO as shown in the inset of Fig. 4c. Therefore, to study the effect of different concentrations of 350 nm Fe 3 O 4 NRs on heating efficiency, the SAR value was measured and analyzed at 308 Oe and 360 KHz. Figure 4d shows the heating performance as a function of temperature for 350 nm Fe 3 O 4 NRs with different concentrations of 0.1, 0.2, and 0.4 mg/mL Fe, where a shorter time of 10 min is observed to easily reach 42 °C for 0.2 mg/mL Fe 3 O 4 NRs, indicating that it is suitable for high temperature magnetothermal application. Further, the heating rate significantly accelerates as the concentration of Fe increases to 0.4 mg/mL. Correspondingly, the sample with 0.2 mg/mL Fe achieves the largest SAR value of 1045 W/g, superior to other concentration samples as shown in Fig. 5a, demonstrating the excellent magnetic hyperthermia performance of Fe 3 O 4 NRs, further providing a powerful basis for the development and utilization of a magnetic hyperthermia agent. The reduction in the heating efficiency with increasing concentration of 0.4 mg/mL Fe maybe due to the higher aggregation of the Fe 3 O 4 NRs, which tends to negatively affect their heating capacity 35,45 . In addition, Fe 3 O 4 NRs have a low cytotoxic effect for gynecological cancer cells with the concentration of Fe from 5 to 100 μg/mL (Fig. 5b), revealing the good biocompatibility of biomedical Fe 3 O 4 NRs. www.nature.com/scientificreports www.nature.com/scientificreports/ Therefore, further study on cell magnetic hyperthermia was performed. All protocols for animal research conformed to the Guide for the Care and Use of Laboratory Animals, and approved by the Lanzhou University Ethics Committee. All the in vivo studies were conducted according to guidelines that were approved by the Institutional Animal Care and Use Committee of the Tsinghua University. After 10 min of magnetic hyperthermia with Fe 3 O 4 NRs-GO, the growth rate of tumors is significantly slowed by tracking the growth of tumors in nude mice for 18 days (Fig. 5c,d). These results indicate that Fe 3 O 4 NRs can be used as a high-performance magnetic hyperthermia agent to effectively limit the growth of gynecological cancer.

conclusions
Highly crystalline and tunable size Fe 3 O 4 NRs have been successfully synthesized by reducing β-FeOOH precursor at 340 °C, while their magnetic and inductive heating properties have been researched. At room temperature, 460, 350, and 250 nm Fe 3 O 4 NRs could display relatively high M s values, where the 350 nm Fe 3 O 4 NRs exhibits a large SAR value of 1045 W/g at low magnetic field (308 Oe), attributing to the high M s and effective anisotropy of nanorod materials. As a result, it reveals good biocompatibility, which is a precondition for magnetic hyperthermia investigation on cancers in the future. Furthermore, as a magnetic hyperthermia agent, the as-prepared Fe 3 O 4 NRs could effectively alleviate the growth of gynecological cancer in the nude mice model, providing an effective approach for the treatment of clinical gynecological cancer in the future.

Data availability
The datasets generated during and/or analyzed during the current research are available from the corresponding author on reasonable request.