Cell lines and animals
B16-F10 melanoma cells were purchased from ATCC (Manassas, VA) and maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified 37°C incubator at 5% CO2.
6-8 week old female C57/BL6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were maintained according to approved institutional IACUC guidelines in the Comparative Medicine Group Facility of Kansas State University. All animal experiments were conducted according to these IACUC guidelines.
Transmission Electron Microscopy
The sizes of the different nanoparticles were determined by using TEM. This was achieved employing a Philips CM-200 TEM instrument operating at 100 kV. 1-2 micrograms of the MNPs were dissolved in anhydrous tetrahydrofuran THF (5 mL) and one drop of the resulting nanoparticle solution was spread over a copper grid (300 mesh size) supporting a thin film of amorphous carbon. To reduce the damage from the electron beam, the sample was cooled to liquid nitrogen temperature during data collection.
Porphyrin-tethered Stealth-Coated (Bi) Magnetic Fe/Fe3O4 Nanoparticles
The synthesis of the stealth-coated dopamine-labeled Fe/Fe3O4 nanoparticles featuring tethered 4-tetracarboxyphenyl porphyrins (TCPP) is reported in a separate publication (Wang et al., ACS Nano, 2010, in preparation). Briefly, Fe/Fe3O4-core/shell nanoparticles were synthesized by NanoScale Corporation and then coated with dopamine-anchored ligands. The structure of the nanoparticles is shown in Figure , and results from electron microscopy are shown in Figure . The diameter of the Fe(0)- core was 5.4 ± 1.1 nm; the diameter of the inorganic Fe/Fe3O4-nanoparticles was determined to be 7.2 ± 2.8 nm. Using the program IMAGE (NIH), we have determined the polydispersity index of the Fe/Fe3O4-nanoparticles to be 1.31. Note that the stealth ligand has a length of 2.5 nm (AM1-Chemdraw Ultra 3D package, Cambridge Soft Corporation, Cambridge, MA 02140), so that the resulting bimagnetic nanoparticles are 12 ± 3 nm in size. The porphyrin-labels have a diameter of 1.95 nm (AM1). Note that the dopamine-anchored tetraethylene glycol ligand (I) and the TCPP-linked dopamineanchored tetraethylene glycol ligand (II) have been synthesized separately. The binding of the ligands to the Fe3O4 layer was achieved in anhydrous THF under argon; the molar ratio of ligands I/II was 100/4. The reaction procedure is described in detail in a separate paper (Wang et al., ACS Nano, 2010, in preparation). We assume a statistical distribution of the ligands at the Fe3O4 surface. Assuming a Poisson distribution, 96.4 percent of the Fe/Fe3O4 NPs at the chosen ratio feature at least one chemically linked TCPP unit, which will act as "bait" for the B16-F10 cancer cells. The solubility of the organically coated Fe/Fe3O4 NPs was determined to be 0.35 mg ml-1 and the Specific Adsorption Rate (SAR) at the field conditions described here was 64 ± 2 Wg-1 (Fe).
Composition of the 4-tetracarboxyphenyl porphyrin (TCPP)-labeled, dopamine-anchored tetraethylene glycol ligands. Nanoparticles of 7.2 nm (outer diameter) require approximately 120 dopamine-anchored ligands (assuming a monolayer).
TEM image of Fe/Fe3O4 core/shell nanoparticles featuring an organic protective dopamine-anchored stealth layer.
Determination of iron concentration in MNPs
Iron concentration in MNPs was measured using the Ferrozine-based spectrophotometric iron estimation method [15
]. For this method, 50 μl of MNPs were diluted to 1 ml with distilled water. MNPs were then lysed by incubating for 2 hours at 65-70°C after the addition of 0.5 ml of 1.2 M HCl and 0.2 ml of 2 M ascorbic acid. After incubation, 0.2 ml of reagent containing 6.5 mM Ferrozine, 13.1 mM neocuproine, 2 M ascorbic acid, and 5 M ammonium acetate was added and incubated for 30 minutes at room temperature. After 30 minutes, the optical density of the samples was measured using a UV-VIS spectrophotometer at 562 nm. A standard curve was prepared using 0, 0.1, 0.2, 0.5, 1, 2, 5 μg/ml ferrous ammonium sulfate samples. Water with all other reagents is used as blank.
The nanoparticles used in these experiments are dominated by Néel relaxation due to the superparamagnetic nature of the iron(0) cores. The hyperthermia apparatus (Superior Induction Company, Pasadena, CA) used here has a "heavy duty" induction heater converted to allow measurement of the temperature change of a sample. In the setup, a remote fiber optic probe (Neoptix, Quebec, Canada) is used to monitor the temperature change. The frequency is fixed (366 kHz, sine wave pattern); field amplitude is 5 kA/m. The coil diameter is 1 inch, 4 turns continuously water cooled. For all in vivo experiments, the mice were placed into the induction coil using a specially designed Teflon supporter so that tumors were located exactly in the region of the AMF possessing the highest field density.
Cytotoxicity of Magnetic Nanoparticles on B16-F10 cells
Potential cytotoxic effects of MNPs were studied by incubating cells in differing concentrations of MNPs. B16-F10 cells were incubated overnight with MNPs amounts corresponding to 5, 10, 15, 20, and 25 μg/mL iron. After incubation, the medium was removed and the cells were washed twice with DMEM and cells were counted via hemocytometer with Trypan blue staining. This method also allows counting non-viable cells since only they allow the blue stain into the cell. All experiments were run in triplicate and repeated at least twice.
Temperature measurements on mice
MNPs containing 100 μg of iron in 100 μl of distilled water were injected into the rear limb muscle of one mouse and the leg was then exposed to AMF for 10 min. An optical temperature probe was inserted intramuscularly at the injection site and the temperature increase was measured during AMF exposure. At the same time, the body temperature was monitored with a separate temperature probe.
Ten mice were transplanted subcutaneously into each rear limb above the stifle with 1 × 106 B16-F10 melanoma cells suspended in PBS. 120 μL of saline were injected into melanomas on the left leg of all mice and 120 μl MNPs containing 1 mg Fe/mL were injected into right leg tumors of all mice in three injections on day 4, 5, 6 (total of 360 μg iron). Because the MNPs were dilute and tumor volumes were quite small, 3 injections instead of 1 were needed in order to reach this level of MNPs. Both left (saline) and right (MNPs) leg tumors of five of the mice were exposed to AMF for 10 minutes soon after injections. Tumors on the remaining five mice were not exposed to AMF. Based on this, there were 4 groups which tested the effects of MNPs with and without AMF and of AMF alone: Group 1: Intratumoral saline injection, not exposed to AMF (left legs of first five mice); Group 2: Intratumoral injection of saline, exposed to AMF (left legs of remaining five mice); Group 3: Intratumoral injection of MNPs, not exposed to AMF (right legs of first five mice); Group 4: Intratumoral injection of MNP, exposed to AMF (right legs of remaining five mice). After three AMF exposures, tumor sizes were measured with a caliper on days 8 to 14, and tumor volume was calculated using the formula 0.5aXb2 (a = longest diameter; b = smaller diameter). After 14 days mice were euthanized, tumors were excised, and tumor weights were measured.
Intravenous administration of MNPs with AMF exposure
On day 0, 0.35 × 106 B16-F10 melanoma cells were injected subcutaneously into the right legs of 27 mice. Mice were randomly divided into three groups: Group 1, IV MNPs, no AMF; Group II, IV MNPs, AMF; Group III, DMEM control, no AMF. On days 6, 9, and 11 after tumor cell transplant, MNPs corresponding to 226 mcg of iron were injected intravenously into each mouse in groups I and II. On the same day, DMEM was injected intravenously into group III. For group II, tumors were exposed to AMF for 10 minutes one day after each I.V. MNPs injection (total of three AMF treatments). MNPs not coated with porphyrins were not sufficiently soluble in water, so we were not able to use them as control. Tumor sizes were measured using a caliper on days 14 and 18, and tumor volume was calculated as described above. On day 18 all mice were euthanized, tumors were excised, and tumor weights were measured.
After euthanizing mice, lung, liver, and tumors were collected and snap frozen. 8-10 μm sections were made in a cryostat (Leitz Kryostat 1720). Staining for iron content on these sections was carried out by using Perl's Prussian blue staining kit (Polysciences, Inc., Warrington, PA). Apoptosis was evaluated using a DeadEnd Fluorometric TUNEL kit. (Promega Corp., Madison, WI) following the manufacturer's instructions.
Statistical analyses were performed by Macanova 4.12 (School of Statistics, University of Minnesota, Minneapolis, MN). The means of the experimental groups were evaluated to confirm that they met the normality assumption. To evaluate the significance of overall differences in tumor volumes and tumor weights between all in vivo groups, statistical analysis was performed by analysis of variance (ANOVA). A p-value less than 0.1 was considered as significant. Following significant ANOVA, post hoc analysis using least significance difference (LSD) was used for multiple comparisons. Significance for post hoc testing was set at p < 0.1. All the tumor volumes and weight data were represented as mean +/- standard error (SE) on graphs.