Preparation of A-MNCs and HA-MRCAs A-MNCs were fabricated using the nano-emulsion method [23]. 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. buy Z-DEVD-FMK 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 selleck 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 P-type ATPase 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 https://www.selleckchem.com/products/mm-102.html reference wavelength of 600 nm.