Polysorbate 80 (polyoxyethylene sorbitan monooleate, P80), spermine, 1,10-carbonyldiimidaziole (CDI), 1,4-dioxane (99.8%), iron(III) acetylacetonate, manganese(II) acetylacetonate, 1,2-hexadecanediol, dodecanoic acid, dodecylamine, benzyl ether, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Hyaluronic acid (20 kDa) was obtained from Lifecore Biomedicals (Chaska, MN, USA). Phosphate-buffered saline (PBS; 10 mM, pH 7.4), Dulbecco's modified Eagle's medium, Roswell Park Memorial Institute medium (RPMI), and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Both MDA-MB-231 and MCF-7 cells, breast carcinoma cell lines [26–28], were obtained from the American Type Culture Collection (Manassas, VA, USA). Sulfo-N-hydroxysuccinimide (sulfo-NHS) and 2,4,6-trinitrobenzene sulfonic acid (TNBSA) solution were purchased from Pierce (Thermo Scientific, Waltham, MA, USA). All other chemicals and reagents were of analytical grade.
Synthesis of MNCs
Monodispered magnetic nanocrystals, soluble in hydrophobic solvent, were synthesized using the thermal decomposition method . First, iron(III) acetylacetonate (2 mmol), manganese (II) acetylacetonate (1 mmol), 1,2-hexadecanediol (10 mmol), dodecanoic acid (6 mmol), and dodecylamine (6 mmol) were dissolved in 20 mL of benzyl ether under a blanket of nitrogen. The mixture was reacted for 2 h at 200°C and then further heated at 300°C for 1 h. All processes were under nitrogen atmosphere. After the mixtures were cooled at room temperature, the products were purified twice with 20 mL of pure ethanol. Finally, MNCs were grown to approximately 12 nm by the seed-mediated growth method.
Preparation of A-MNCs and HA-MRCAs
A-MNCs were fabricated using the nano-emulsion method . First, 10 mg of MNCs was dissolved in 4 mL of n-hexane (organic phase). The organic phase was injected into 30 mL of de-ionized water (aqueous phase) containing 100 mg of aminated P80. After mutual saturation, the solution was emulsified for 20 min under ultrasonification (ULH700S, Ulssohitech, Cheongwon-gun, South Korea) at 450 W. The mixture was kept overnight at room temperature to remove the volatile organic solvent. The products were purified using a centrifugal filter (Centriprep YM-3, 3-kDa molecular weight cutoff (MWCO), Amicon, Millipore Corporation, Billerica, MA, USA) in triplicate at 3,000 rpm for 30 min.
HA-MRCAs with different molar ratios of HA were fabricated by EDC-NHS chemistry. First, the pH of the A-MNC solution was adjusted to neutral condition by the addition of 0.1 N HCl solution. Then, various amounts of HA (0.43, 1.7, and 6.8 μmol) were dissolved in the 40 mL of de-ionized water followed by the addition of EDC and sulfo-NHS. Each HA solution was added to A-MNC solution containing 5 mg of MNCs. The HA and A-MNCs were reacted for 2 h at room temperature. Finally, EDC, sulfo-NHS, and unbound HA were removed using dialysis (MWCO, 25, 000) against excess de-ionized water.
Characterization of A-MNCs and HA-MRCAs
The size distributions and zeta potential values of A-MNCs and HA-MRCAs were measured using laser scattering (ELS-Z, Otsuka Electronics, Osaka, Japan). The inorganic ratios (%) and the crystallinities of magnetic nanocrystals in A-MNCs and HA-MRCAs were analyzed using a thermo-gravimetric analyzer (SDT-Q600, TA Instruments, Newcastle, DE, USA) and X-ray diffraction (X-ray diffractometer Ultima3, Rigaku, Tokyo, Japan) at 25°C, respectively. The magnetic properties of A-MNCs and HA-MRCAs were also detected by a vibration sample magnetometer (model 9407, Lake Shore Cryotronics, Inc., Westerville, OH, USA) at 25°C.
Cell viability assay for A-MNCs and HA-MRCAs
The cytotoxic effect of A-MNCs and HA-MRCAs against MDA-MB-231 cells (CD44-abundant cancer cell line) was analyzed by measuring the inhibition of cell growth using an assay for WST-1 ((4-(3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate)). MDA-MB-231 cells were maintained in RPMI containing 10% FBS and 1% antibiotics at 37°C in a humidified atmosphere with 5% CO2. MDA-MB-231 cells were harvested at a density of 1.0 × 104 cells/100 μL in a 96-well plate and incubated at 37°C in 5% CO2 atmosphere overnight. The cells were then treated with various concentrations of A-MNCs and HA-MRCAs for 24 h. After incubation, the cells were rinsed with 100 μL PBS (pH 7.4, 1 mM), and then 10 μL of WST-1 solution was added to each well. The absorbance was measured at 450 nm with a reference wavelength of 600 nm. The relative percentage of cell viability was determined as the ratio of formazan intensity in viable cells treated with A-MNCs and HA-MRCAs to the intensity in non-treated (control) cells [29–31].
Cellular targeting efficiency of HA-MRCAs
The targeting efficiency of HA-MRCAs was examined by MR imaging of breast carcinoma cell line MDA-MB-231 cells (high CD44 expression) and MCF-7 cells (low CD44 expression). First, target cells (1.0 × 107 cells) were harvested and washed three times with blocking buffer (FBS (0.2%) and NaN3 (0.02%) in phosphate-buffered solution (pH 7.4, 10 mM)) to inhibit non-specific binding effects. The solutions containing HA-MRCAs were applied to each cell line (1 and 0.5 μg, respectively) at 4°C for 30 min. The cells were then washed with blocking buffer three times to remove non-binding HA-MRCAs. Next, 200 μL of 4% paraformaldehyde was added to re-suspend the cells. After targeting efficiency was analyzed via MRI, the cells were dissolved in nitric acid for 2 h at 180°C, and the concentrations of magnetic nanocrystals (Fe + Mn) were measured using inductively coupled plasma atomic emission spectrometry (ICP-AES).
MR imaging procedures
We performed in vitro MR imaging experiments with a 1.5-T clinical MRI instrument with a micro-47 surface coil (Intera, Philips Medical Systems, Best, The Netherlands). The T 2 weights of the A-MNC- and HA-MRCA-treated cells (MDA-MB-231 and MCF-7 cells) were measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence at room temperature with the following parameters: TR = 10 s, 32 echoes with 12-ms even echo space, number of acquisitions = 1, point resolution of 156 × 156 μm, and section thickness of 0.6 mm. For acquisition of T 2-weighted MR images of A-MNC- and HA-MRCA-treated cells, the following parameters were adopted: resolution of 234 × 234 μm, section thickness of 2.0 mm, TE = 60 ms, TR = 4,000 ms, and number of acquisitions = 1.