The MNPs were from Shanghai Jiaotong Unversity. Iron chloride hexahydrate was from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The MNPs were coated with cetyltrimethyl ammonium bromide at the size of 25 to 35 nm. Magnetic field (MagneSphere Technology Magnetic Separation Stands, Z5341) was from Promega (Madison, WI, USA). Muffle furnace (SX-8-10) was from Tianjin Taisite Instrument Co. Ltd. (Tianjin City, China).
Establishing a method to determine ferric ions in vitro
For quantitive analysis of MNPs the concentration of iron ion was created as a standard for MNPs. Ferric chloride was use to analyze the iron content. Potassium thiocyanide colorimetry showed a good linear relationship and was used to determine ferric ions in MNPs treated with hydrochloride acid at 100°C. Different concentration of 0.5 ml ferric chloride was mixed with an equal volume of 2N hydrochloride acid and boiled for 10 min. The acidified solution was cooled to room temperature and added 120 μl of 5M potassium thiocyanide. Ninety-six-well plates were used in this detection method. The colored reaction product (150 μl/well) was measured at 480 nm on a spectral scanning multimode reader (Varioskan Flash version 2.4.3, Thermo Scientific, Logan, UT, USA). The sonicated and acidified MNPs could be treated in the same way and its ferric ions concentration could be calculated by the ferric chloride standard curve.
The influence of animal tissue on the determination of ferric ions in MNPs
For the assessment of MNPs in different tissues by colorimetry, the normal mice were narcotized with ether. The whole blood was collected into heparin-coated tube and diluted with nine volumes of saline before mixing with MNPs. Different tissues were harvested. Tissues from three mice were mixed and homogenized in ten volumes of ice-cold saline in a homogenizer (ULTRA-TURRAX T25, IKA-Labortechnik, Staufen, Germany) for 10 s. Three hundred microliters of MNPs at 0.078 to 40 μg/ml in saline was mixed with an equal volume of tissue homogenate and acidified with 80 μl of 6N hydrochloric acid. The acidified solution was centrifuged at 14,000 rpm for 6 min, and then, the supernatants were colorized with 80 μl of 5M potassium thiocyanide.
The iron background in mouse tissues
One hundred microliters of 10% mouse tissue homogenate was mixed with 500 μl of saline and then treated with proteinase K at the final concentration of 100 μg/ml under 55°C to 65°C for 0.5 h. The samples were then transferred into crucibles, oven dried on a hot plate, and then carbonized in a muffle at 420°C for 2 h. Cooled to room temperature, the samples were acidified with 0.5 ml 1N hydrochloric acid at 100°C for 10 min. Cooled to room temperature, 1N hydrochloric acid was replenished to the final volume of 1.0 ml. Centrifuged at 10,000 rpm for 10 min, 0.5 ml of the supernatants was colorized with 60 μl of 5M potassium thiocyanide. The concentration of ferric ions was calculated referencing the result from ferric chloride standard.
The determination of MNPs in blood treated with proteinase K with/without magnetic field collection
MNPs at different concentrations in 300 μl of saline were mixed with 30 or 300 μl of mouse whole blood and replenished with saline to the final volume of 600 μl. Aliquots were treated with/without 3 μl of proteinase K, 20 mg/ml, to the final concentration of 100 μg/ml under 55°C to 65°C for 0.5h and centrifuged at 14,000 rpm for 5 min. The 200 μl of the supernatant was mixed with 400 μl of saline to check whether Fe3O4 remained after centrifugation. The precipitate was washed with 0.6 ml saline, centrifuged two times and suspended in 600 μl of saline. Each sample was added with 80 μl 6N hydrochloric acid and boiled for 10 min. After cooling in a water bath at room temperature, the solutions were colorized with 80 μl 5M KSCN, and their A480 were measured.
Using magnetic field to separate MNPs from the endogenous iron followed by carbonation to ruin ferric ions binding groups
Being a standard, 300 μl of MNPs at 40 μg/ml in saline was diluted with 200 μl of saline, acidified with 500 μl 2N hydrochloric acid, and colorized with 120 μl 5M KSCN. The same aliquots of MNPs, 300 μl in saline, were respectively mixed with 200 μl of saline and 200 μl of saline containing 50% mouse blood. The crucibles containing these solutions were put on a magnetic field, and the MNPs were collected and washed two times with saline. The precipitates could be directly acidified with 1 ml 1N hydrochloric acid or carbonized in a muffle furnace at 420°C for 2 h, cooled to room temperature, and then be acidified. After acidification at 100°C for 10 min and cooling to room temperature, all the samples above were replenished and colorized with 120 μl 5M KSCN. Using the same procedure, MNPs mixed in the mouse tissue homogenate was collected and determined. The collected ratio was calculated and compared with the result of the MNPs in saline.
Determination of MNPs in the blood of mice treated with MNPs by intravenous injection
CD-1 mice were i.v.-administrated with MNPs at a dose of 7.5 mg/kg. At different time points after MNP treatment, the whole blood was collected in heparin-coated tube. In a crucible, 0.1 ml of whole blood was mixed with 0.9 ml of saline, and MNPs were collected and washed two times with saline accompanied with magnetic field separation. The collected precipitates were carbonized as before. The residues were suspended in 0.5 ml 1N hydrochloric acid and colorized with 60 μl 5M KSCN. The concentration of ferric ions was calculated, referencing with ferric chloride standard with attention on the volume used.