Assembly of Iron Oxide Nanocubes for Enhanced Cancer Hyperthermia and Magnetic Resonance Imaging

Multiple formulations of iron oxide nanoparticles (IONPs) have been proposed for enhancing contrast in magnetic resonance imaging (MRI) and for increasing efficacy in thermal ablation therapies. However, insufficient accumulation at the disease site and low magnetic performance hamper the clinical application of IONPs. Here, 20 nm iron oxide nanocubes were assembled into larger nanoconstructs externally stabilized by a serum albumin coating. The resulting assemblies of nanocubes (ANCs) had an average diameter of 100 nm and exhibited transverse relaxivity (r2 = 678.9 ± 29.0 mM‒1·s‒1 at 1.41 T) and heating efficiency (specific absorption rate of 109.8 ± 12.8 W·g‒1 at 512 kHz and 10 kA·m‒1). In mice bearing glioblastoma multiforme tumors, Cy5.5-labeled ANCs allowed visualization of malignant masses via both near infrared fluorescent and magnetic resonance imaging. Also, upon systemic administration of ANCs (5 mgFe·kg‒1), 30 min of daily exposure to alternating magnetic fields for three consecutive days was sufficient to halt tumor progression. This study demonstrates that intravascular administration of ANCs can effectively visualize and treat neoplastic masses.

Transmission electron microscopy (TEM). The TEM images were produced by a JEOL 2,100 field emission gun TEM operating at 200 kV with a single tilt holder using ultrathin carbon type-A 400 mesh copper grids (Ted Pella Inc.). The average sizes and size variations were obtained by counting more than 1,000 particles using Image-Pro Plus 5.0 (Media Cybernetics, Inc., Silver Spring, MD).
Dynamic light scattering (DLS) and zeta potential analysis. All water-soluble ANC suspensions were analyzed by DLS and zeta potential to measure average hydrodynamic size (nm) and surface charge (mV), respectively, using a ZEN-3,600 Zetasizer Nano (Malvern, UK) equipped with a HeNe 633 nm laser. The average hydrodynamic size (nm) was calculated as the mean size of the first peak in the number distribution. Standard deviations of hydrodynamic size (nm) and zeta potential (mV) were calculated from five replicate measurements.

Inductively coupled plasma optical emission spectroscopy (ICP-OES).
The concentration of iron in ANC suspension was measured by a Perkin Elmer ICP-AES instrument equipped with an auto sampler. Purified ANC solutions were acid digested using HNO3 (70 %) and H2O2 (30 %) and diluted with deionized water for ICP analysis.
Magnetic resonance relaxivity measurement. To measure the relaxivities r1 and r2 of ANCs, a magnetic resonance relaxometer was used at 1.41 T (NMR analyzer, mq 60, Bruker). Various concentrations of ANC solution were prepared (0.014 ÷ 0.35 mM for r1 and 0.0014 ÷ 0.022 mM for r2), and the relaxivity values were calculated by the ratio of 1/T1,2 and concentration (mM) of Fe. The r1 and r2 were calculated using Equation 1 [3]: where T1,2 represents the relaxation time of the sample, Twater is that for water, r1,2 (mM -1 s -1 ) are the relaxivity values, and [Fe] is the concentration of Fe.
Specific absorption rate measurements for hyperthermia treatment. The magnetic heating property of ANCs was measured by a custom-built hyperthermia system. The measurement was performed using a radio frequency generator producing an alternating magnetic field (AMF) with a frequency (f) of 512 KHz and field amplitude (H) of 10 kA m -1 [4]. A cooling system thermally isolated the vial from the high temperature of the coil when applying AMF. ANC suspension (600 μL) was placed in a cylindrical probe (4 mm ID x 40 mm height), and the temperature was monitored with an optical probe (OptiSens Instrument) immersed into the geometrical center of the solution. When the sample suspension reached the equilibrium temperature (~ 19 °C), the magnetic field was activated, and the temperature was recorded every second for about 15-20 minutes. The specific absorption rate (SAR) was calculated based on Equation 2 [5]: where T is the temperature of the nanocube suspension, t is the time, Cp is the heat capacity of the buffer, and mFe is the final mass fraction of the iron in the sample suspension.

Superconducting Quantum Interference Device characterization (SQUID).
To determine the magnetic behavior and saturated magnetization value of iron oxide NCs, the magnetization measurement was carried out with a Quantum Design SQUID magnetometer MPMS-XL. The sample was prepared by injecting a certain amount of nanocube suspension (300 μL, 3,000~5,000 mg L -1 ) into an NMR tube. The tube was then sealed under vacuum and placed into a plastic straw for measurements. DC magnetic measurement was performed at 300 K in a 0-50 Oe field. Magnetization and hysteresis curves were collected in the range from -50 KOe to 50 KOe at 300 K.