Synthesis of Fe/Fe3O4-nanoparticles
The synthesis of Fe/Fe3O4-NPs is proprietary information of NanoScale Corporation, Manhattan, KS.
Porphyrin-tethered Stealth-Coated (Bi) Magnetic Fe/Fe3O4 Nanoparticles
Aminosiloxane-coated Fe/Fe3O4-core/shell nanoparticles were synthesized by NanoScale Corporation (Manhattan, KS); the synthesis is represented in .
Synthesis of Fe/Fe3O4/ASOX
The synthesis of ASOX-covered Fe/Fe3
-NPs was performed by adapting a procedure from the literature.63
Twenty mg Fe/Fe3
nanoparticles were suspended in 10 mL anhydrous tetrahydrofuran (THF). After sonicating, the undissolved solid (< 1 mg) was separated by precipitation through low-speed centrifugation (1500 RPM, 5 min.). The clear solution was transferred to another test tube and 3-aminopropyltriethoxylsilane (see ) was added to the solution. After sonicating, the nanoparticles were collected by high speed centrifugation (15,000 RPM for 15 min). After re-dispersion and subsequent collection in THF, the Fe/Fe3
/ASOX-NPs (7.5 mg) were collected, dried in high vacuum, and stored under argon.
Synthesis of stealth-coated Fe/Fe3O4/ASOX-nanoparticles
Forty mg dopamine-based ligand (see ) was dissolved in 5.0 mL THF, 20 mg Fe/Fe3O4/ASOX nanoparticles and 1.0g CDI (carbonyl-di-imidazole) were added as solids. After sonicating, the nanoparticles were collected by high speed centrifugation (15,000 RPM for 15 min). After re-dispersion and subsequent collection in THF, the Fe/Fe3O4/stealth-NPs (15 mg) were collected, dried in high vacuum, and stored under argon.
Synthesis of TCPP-linked stealth-coated Fe/Fe3O4/ASOX-nanoparticles
2.5 mg of TCPP (Tetrakis(4-carboxyphenyl)porphyrin) was dissolved in 5.0 mL THF; 20 mg Fe/Fe3
/ASOX/stealth nanoparticles and 1.0/0.05g EDC/HOBT (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/1-hydroxybenzo-triazole) were added as solids.64
After sonicating, the nanoparticles were collected by high speed centrifugation (15,000 RPM for 15 min). After re-dispersion and subsequent collection in THF, the TCPP-labeled Fe/Fe3
/ASOX/stealth-NPs (13.5 mg) were collected, dried in high vacuum, and stored under argon. We have determined by using UV/Vis-spectroscopy (λabs
(TCPP) = 416nm, = 365,000 M−1
) that 5±0.5 TCPP units are bound to one stealth-coated Fe/Fe3
/ASOX nanoparticle, on average.
As already stated, HRTEM performed at the University of Kansas Microscopy and Analytical Imaging Laboratories has revealed that the nanoparticles are composed of nano-rods (5–10nm in length, 1–4nm in diameter). These nano-rods form clusters of 16.0±1.5 nm in diameter. The thickness of the aminosiloxane shell that is surrounding the whole Fe/Fe3
-clusters is 2.0±0.4nm. This is consistent with an average diameter of the Fe/Fe3
/ASOX-nanoparticles of 20±2.3nm. Using the program IMAGE (NIH), we have determined the polydispersity index of the Fe/Fe3
/ASOX nanoparticles to be 1.15. Note that the stealth ligand has a length of 2.5 nm (AM1- Chemdraw Ultra 3D package, Cambridge Soft Corporation, Cambridge, MA), so that the resulting Fe/Fe3
/ASOX stealth nanoparticles are 25±2.3 nm in size. The space demand for the dopamine anchor is 1.094nm2
(AM1). One Fe/Fe3
/ASOX nanoparticle of 20nm in diameter can bind 1150 organic ligands. The porphyrin-labels have a diameter of 1.95 nm (AM1). The binding of the ligands to the terminal amino groups of the aminosiloxane layer was achieved in THF under argon in the presence of CDI as coupling reagent; the molar ratio of ligands L1/L1-TCPP was 1000/3.5. We assume a statistical distribution of the ligands at the surface. Assuming a Poisson distribution,65
99.33 percent of the Fe/Fe3
/ASOX/stealth NPs at the chosen ratio (5 TCPP units per nanoparticle) feature at least one chemically linked TCPP unit. The solubility of the organically coated Fe/Fe3
NPs was determined to be 2.25 mg ml−1
and the Specific Adsorption Rate (SAR) at the field conditions described here was 620±30 Wg−1
(Fe). We have determined the zeta-potential of the Fe/Fe3
/ASOX/stealth-TCPP nanoparticles by using Zeta Plus (Brookhaven Instruments, Holtsville, NY) to be 34mV in 0.1M PBS-buffer at 298K; their BET-surface was determined in NanoScale Corporation’s analytical laboratory to be 72±2 m2
Tissue culture of C17.2 neural progenitor cells and B16-F10 melanoma cells
B16-F10 melanoma cells were purchased from ATCC (Manassas, VA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO) and 1 % penicillin- streptomycin (Invitrogen) at 37 °C in a humidified atmosphere containing 5% carbon dioxide.
C17.2 neural progenitor cells (NPCs) were a gift from V Ourednik (Iowa State University. Originally developed in Evan Snyder’s lab,66
these cells were maintained in DMEM supplemented with 10% FBS (Sigma-Aldrich), 5% horse serum (Invitrogen), 1% glutamine (Invitrogen), and 1% penicillin- streptomycin (Invitrogen).
Cytotoxicity of MNPs on neural progenitor cells and B16-F10 cells
Potential cytotoxic effects of MNPs (NanoScale Corporation, Manhattan, KS) were studied by incubating C17.2 NPCs and B16-F10 melanoma cells in different concentrations of MNPs (as determined by iron content). NPCs and B16-F10 cells were plated at 50,000 cells/cm2
and incubated overnight with their respective media containing MNPs at concentrations of 5, 10, 15, 20, or 25 µg/mL iron. After incubation, medium was removed and cells were washed twice with DMEM. Cells were lifted via
trypsinization, and live and dead cell numbers were counted via
hemocytometer with Trypan blue staining. This method allows counting of viable (colorless) and non viable (blue stained) cells, since only the dead cells allow the blue stain into the cell. A photograph of Trypan-blue stained cells showing the blue coloration of dead cells in the hemocytometer grid is shown in Supplemental Figure S2 (Supporting Information)
. NPCs and B16-F10 cells were used in three separate trials and each experiment was done in triplicate.
Prussian blue staining on NPCs
The loading efficiency of MNPs into NPCs was assessed using Perl’s Prussian Blue stain kit (Polysciences, Inc., Warrington, PA). After overnight incubation in NPC medium containing 25 µg/mL iron in MNPs, NPCs were washed twice with DMEM and PBS and fixed with 4% glutaraldehyde for 10 min. Fixed NPCs were incubated in a solution containing equal amounts of 4% potassium ferrocyanide and 4% HCl for 20 minutes. After 20 min incubation, NPCs were washed twice with 1× PBS and counterstained with nuclear fast red solution for 30 minutes. Images were captured using a Zeiss Axiovert 40 CFL microscope (New York) and a Jenoptik ProgRes C3 camera (Jena, Germany).
Loading strategy of MNPs and determination of iron amounts
The loading efficiency of NPCs with various iron concentrations of MNPs was determined spectrophotometrically using a Ferrozine iron estimation method.67
For this method, cells were incubated overnight with NPC medium containing different concentrations of MNPs and then washed twice with DMEM and 1× PBS. Cells in medium without MNPs were used as controls. All NPCs (control cells and cells loaded with various iron concentration of MNPs) were trypsinized, counted, centrifuged, and total cells were resuspended in 2 mL distilled water. Cells were then lysed by adding 0.5 mL of 1.2 M HCl and 0.2 mL of 2M ascorbic acid and incubating at 65–70 °C for 2 hours. After 2 hours, 0.2 mL of reagent containing 6.5 mM Ferrozine (HACH, Loveland, CO), 13.1 mM neocuproine (Sigma-Aldrich, St Louis, MO), 2 M ascorbic acid (Alfa Aesar, Ward Hill, MA) and 5 M ammonium acetate (Sigma-Aldrich, St Louis, MO) was added and incubated for 30 minutes at room temperature. After 30 minutes, samples were centrifuged at 1000 rpm for 5 min, and supernatant optical density was measured by UV-VIS spectrophotometer (Shimadzu, Columbia, MD) at 562 nm. A standard curve was prepared using 0, 0.1, 0.2, 0.5, 1, 2, and 5 µg/mL ferrous ammonium sulfate samples. Water with all other reagents was used as a blank. From the standard curve, iron concentration in cell samples was determined. Iron concentration per single cell was estimated by dividing the iron amount in each cell sample by the total number of cells in that sample.
AMF-induced temperature changes in vitro
To verify the temperature increase by NPCs loaded with MNPs in a simulated tumor environment, NPCs were loaded overnight with MNPs equivalent to 15 µg/mL Fe. After incubation, cells were washed twice with DMEM and twice with 1× PBS to remove free MNPs. Cells were lifted with 0.1% trypsin-EDTA, and 1×106 cells were pelleted by centrifugation in 2 mL centrifuge tubes. 1.5 ml of 4% agarose solution was added on top of the cell pellet to mimic the extracellular matrix in tumor tissues. Agarose centrifuge tubes containing pelleted NPCs without MNPs were used as negative controls and were made as described above. The experiment was conducted in triplicate. Before each tube was exposed to AMF, two optical probes (Neoptix, Quebec, Canada) were inserted into the tube: one at the pellet, and the second one at the middle of the agarose. Tubes were exposed to AMF for 10 min, and the temperature difference over time was measured by the probes.
Evaluation of selective engraftment of NPCs and magnetic hyperthermia
Female, 6–8 week old, C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were held for 1 week after arrival to allow them to acclimate. Mice were maintained according to approved IACUC guidelines in the Comparative Medicine Group facility of Kansas State University. All animal experiments were conducted according to these IACUC guidelines. On day 0, 3.5 × 105 B16-F10 melanoma cells were injected subcutaneously into 21 C57BL/6 mice, and the mice were randomly divided into three groups. On day 5, 1 × 106 NPCs loaded with MNPs at 20 µg/mL iron concentration were injected intravenously to two groups (NPC-MNP, group I and NPC-MNP+AMF, group II); simultaneously, saline was injected into group III. On the 9th, 10th, and 11th days after tumor inoculation, group II mice with NPC loaded MNPs were exposed to AMF for 10 min daily using an alternating magnetic field apparatus (Superior Induction Company, Pasadena, CA). The frequency is fixed (366 kHz, sine wave pattern); field amplitude is 5 kA/m. Tumor volumes were measured using a caliper on days 8, 10, and 12; they were calculated using the formula 0.5a×b2, where a is the larger diameter and b the smaller diameter of the tumor. All the mice were then euthanized on day 15 and the tissues were collected. The experiment was repeated once with similar results.
All mice were sacrificed 15 days after tumor inoculation by CO2 inhalation and cervical dislocation. Tumor, lung, liver, and spleen were snap-frozen in liquid nitrogen for histological analysis. Tissues were sectioned on a cryostat (Leitz Kryostat 1720, Germany) at 8–10 µm and used for histological studies. Prussian blue staining was performed on these sections using Perl’s Prussian blue stain kit to identify NPCs loaded with MNPs. Apoptotic cell detection in the tissue sections was determined using the DeadEnd fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System (Promega Corporation, Madison, WI), as per the manufacturer’s protocol.
Protein preparation for 2-Dimensional electrophoresis (2-DE)
To identify protein expression differences between tumors from mice receiving AMF after IV saline injection or after IV NPC-MNP injection, 3.5 × 105 B16F10 melanoma cells were injected subcutaneously into two mice. On day 5, 1 × 106 NPCs loaded with MNPs at 20 µg/mL iron concentration were injected intravenously to one mouse; simultaneously saline was injected to the other mouse. On days 9, 10, and 11, tumors were exposed to AMF. On day 15 mice were euthanized and tumors were collected immediately after AMF exposure.
Total protein was prepared from the tumors for use in two-dimensional gel electrophoresis (2-DE) analysis. The following protein isolation protocol was used.68
Briefly, melanoma tissues were homogenized using a Pellet Pestle Mortar (KONTES, Vineland, NJ) in the presence of 0.5 mL of lysis buffer (8 M urea, 2 M thiourea, 4% 3-cholamidopropyl-dimethylammonio-1-propanesulfonate (CHAPS), 100 mM dithiothreitol (DTT), 25 mM Tris-Cl, and 0.2% ampholyte (pH 3 to 10) (Amersham Pharmacia Biotech, Piscataway, NJ). The supernatant was collected and then precipitated using 2 volumes of ice-cold acetone. The final protein pellet was dissolved in 100 µl of the sample buffer (8 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 25 mM Tris-Cl, and 0.2% ampholyte (pH 3 to 10)). Protein concentrations were determined using a reducing agent-compatible and detergent-compatible protein assay kit (Bio-Rad, Hercules, CA).
Fifty micrograms of total protein was resolved at 20°C in the first dimension by isoelectric focusing (IEF) in an IEF cell system (Bio-Rad, Hercules, California) using 7-cm long, pH 3 to 10, precast immobilized pH gradient strips (Bio-Rad). The IEF parameters were 250 V for 15 min, followed by 4,000 V for 5 hrs. At the end of the IEF, the strips were equilibrated sequentially for 10 min each in 1 ml of equilibration buffer I (375 mM Tris-HCl [pH 8.8], 6 M urea, 2% sodium dodecyl sulfate (SDS), 2% DTT) and buffer II (375 mM Tris-HCl [pH 8.8], 6 M urea, 2% SDS, 2.72 mg of iodoacetamide/ml, 0.001% bromophenol blue). Subsequently, second-dimension SDS-polyacrylamide gel electrophoresis analysis was performed on the strips in a Mini-PROTEAN Tetra system by using 12% polyacrylamide gels (Bio-Rad) for 40 min at 200 V at room temperature in a 50 mM Tris-glycine buffer. The 2-DE resolved gels were stained by using a Biosafe Coomassie G-250 kit (Bio-Rad). Coomassie-stained gels were digitalized by using an HP Scanjet 7400c scanner (Hewlett-Packard, Houston, Texas).
Matrix-assisted laser desorption ionization (MALDI)–TOF MS analysis of melanoma tissues treated with AMF and AMF with NSC-MNP proteins
After resolution by 2-DE, proteins from melanomas from mice treated with AMF and MNP-NPC+AMF were picked individually from Coomassie blue stained gels using PROTEINEER spII with sp-Control 3.0 software (Bruker Daltonics, Bremen, Germany) according to the manufacturer’s protocol. Coomassie blue-stained proteins were digested as described by Shevchenko et al
An aliquot of in-gel-digested solution was mixed with an equal volume of a saturated solution of 1ul of 2,5-Dihydroxybenzoic acid (DHB) in 50% aqueous acetonitrile, and 1 ul of mixture was spotted onto a target plate. Protein analysis was performed with a Bruker UltraFlex II MALDITOF using MTP AnchorChip with 384 matrix spots. MALDI-TOF spectra were externally calibrated using a combination of nine standard peptides: bradykinin 1–7 (757.39 Da), angiotensin II (1,046.54 Da), angiotensin I (1,296.68 Da), neurotensin (1,672.91 Da), renin substrate (1,758.93 Da), ACTH clip 1–17 (2,093.08 Da), ACTH clip 18–39 (2,465.19 Da), ACTH clip 1–24 (2,932.58 Da), and ACTH clip 7–38 (3,657.92 Da), spotted onto positions adjacent to the samples. Protein identification was carried out by automatic comparison of experimentally generated monoisotopic values of peptides using MASCOT with a tolerance of 0.5 −0.3 Da and 0 −1missed cleavage, and oxidation of methionine was allowed.
Statistical analyses were performed using WinSTAT (A-Prompt Corporation, Lehigh Valley, PA). 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 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 < 0.05. All the tumor volume data are represented as mean +/− standard error (SE) on graphs.