Treatment of oral cancer using magnetized paclitaxel

N,N’-Bis(salicylidene)ethylenediamine iron (Fe(Salen)) is an anti-cancer agent with intrinsic magnetic property. Here, we covalently linked Fe(Salen) to paclitaxel (PTX), a widely used anti-cancer drug, to obtain a magnetized paclitaxel conjugate (M-PTX), which exhibited magnetic characteristics for magnet-guided drug delivery and MRI visualization. M-PTX increased apoptosis and G2/M arrest of cultured human oral cancer cell lines in the same manner as PTX. Furthermore, marked contrast intensity was obtained in magnetic resonance imaging (MRI) of M-PTX. In a mouse oral cancer model, a permanent magnet placed on the body surface adjacent to the tumor resulted in distinct accumulation of M-PTX, and the anti-cancer effect was greater than that of M-PTX without the magnet. We believe that this strategy may improve future cancer chemotherapy by providing conventional anti-cancer drugs with novel functionalities such as magnet-guided drug delivery or MRI-based visualization/quantitation of drug distribution.


N,N'-Bis(5-tert-butoxycarbonylamino-2hydroxybenzylidene)ethylenediamine (6)
A solution of 5 (260 mg, 1.1 mmol) in anhydrous EtOH (10 mL) was heated to reflux and then ethylenediamine (33 mg, 0.55 mmol) in anhydrous EtOH (10 mL) was added dropwise to the hot solution. After the addition, the mixture was stirred at reflux for 0.5 h. The precipitate was collected by filtration, washed with EtOH (50 mL), and dried in vacuo to give 200 mg of Schiff base ligand 6 as pale yellow needles. 1

Dynamic light scattering (DLS)
The hydrodynamic size and colloidal stability of the M-PTX NPs were determined by dynamic light scattering (DLS) and zeta potential analysis using a Zetasizer Nano ZSP (Malvern Instruments, Ltd., Malvern, UK). Both DLS and zeta potential data were obtained from twelve runs per measurement and exhibited a uniform distribution between runs, which is a criterion of high quality.

Powder X-ray diffraction (XRD) characterization of M-PTX
To prepare a pure M-PTX sample for XRD analysis, 20 mg of M-PTX powder was dissolved in 50 mL of methanol. The solution was stirred thoroughly, then sonicated, and purified by magnetic separation to ensure removal of any undissolved sediment. The resulting clear brown solution was filtered and the filtrate was evaporated in vacuo at room temperature. The XRD measurements were performed with a Rigaku Smart Lab horizontal X-ray diffraction apparatus with a Cu target (output: 45 kV-200 mA). Fe(Salen) powder (TCI, Tokyo, Japan) was used as a standard for XRD.

Real-time cell growth assay
In vitro cell proliferation was measured using a xCELLigence (ACEA Biosciences, California, USA) realtime cellular analysis system [8]. Briefly, the background impedance was measured following the addition of 100 µL of growth medium to 16-well E-plates. Cell suspension containing 5.0 × 10 3 OSC-19 or HSC-3 cells was seeded into the wells. Attachment and proliferation were monitored using the xCELLigence system. When cells reached the logarithmic growth phase, they were further treated with M-PTX or PTX and continuously monitored for ~100 hours. Impedance change, expressed as cell index (CI), was automatically calculated as live cells interacted with the electrodes in the E-plates, as a parameter of viability or cytotoxicity. Growth curves were normalized to the CI at 48 hours before the anti-cancer effects of M-PTX and PTX became apparent.

In vivo MR imaging of M-PTX or PTX after administration
Nine female BALB/c nude mice (Japan SLC, Shizuoka, Japan) were used for all in vivo studies; for evaluation of the accumulation of M-PTX in tumor, five received M-PTX and four were used as the PTX control. The mice were maintained in accordance with the guidelines of the National Institute of Radiological Sciences (NIRS, QST), and all experiments were reviewed and approved by the institute's committee for care and use of laboratory animals.
OSC-19 human carcinoma cells were obtained from the RIKEN BioResource Center (Tukuba, Japan). The cells were maintained in Dulbecco's modified Eagle's medium (D5796, Sigma-Aldrich, Missouri, USA) supplemented with 10 % fetal bovine serum, and incubated in a humidified atmosphere of 5 % CO 2 in air at 37° C. After suspension in phosphate-buffered saline, the cells were subcutaneously grafted (1.0 × 10 6 cells/50 µl) into the femur area of the mice. The tumors were allowed to grow to 5 mm diameter (Day 0) after tumor cell transplantation.
To evaluate M-PTX accumulation in tumors in vivo, M-PTX or PTX was administered three times, once per day, at Days 1, 2 and 3. A permanent magnet was attached at the tumor site for all 3 days. M-PTX or PTX was intravenously administered from a tail vein (80 mg/kg). During Days 0 to 3, there was no significant difference in body weight between M-PTX (n = 5) and PTX (n = 4) administered mice (p > 0.05, 2-way ANOVA with Bonferroni post-hoc tests).
On Day 4 (3 days after the initial administration of the M-PTX or PTX), 2D spin-echo T1-weighted, 2D multi spin-echo T2-weighted and gradient-echo T2*weighted images were acquired. During the in vivo MRI experiments, the rectal temperature of the mice was monitored with an optical fiber thermometer (FOT-L, FISO Technology, Quebec, CA) and maintained at approximately 36.5 ± 0.5° C by warm air flow provided by a homemade automatic heating system based on an electric temperature controller (E5CN, Omron, Kyoto, Japan). The mice were anesthetized with 1.5-2.0% isoflurane (Escain, Mylan, Tokyo, Japan) gas in a 1:5 O 2 :room-air mixture.
T1-weighted images were acquired using a multispin-echo sequence with the following parameters: TR/ TE = 350/9.

Intravenous injection of M-PTX NPs in a mouse model implanted with human oral cancer
OSC-19 cells that had been transfected with luciferase-encoding vector were implanted into the back of Balb/c Slc-nu/nu mice (4-5 weeks old; 4 mice/group) (SLC, Shizuoka, Japan) to create a human oral cancer model. The tumors were allowed to grow to a size of 3-5 mm, and then M-PTX or PTX (12 mg/kg per mouse) were injected daily via a tail vein for 7 days. The tumor volume ratio was calculated by dividing the volume of each tumor by the baseline volume every other day. After 14 days, tumors were harvested, and pathologically examined by hematoxylin-eosin staining (HE) and chemical iron staining.

Magnetically guided focal delivery enhanced the anti-cancer effect of M-PTX in vivo
The photon flux was measured once a week for 19 days, as previously described [9]. Regression rate was calculated using the following formula: Regression rate (%) = Photons/Photons (day0) × 100 Supplementary Table 1 Binding energy was calculated as described [11]. As lower binding energy corresponds to greater stability, the order of stability is M-