2.1 Materials
Iron(III) acetylacetonate, manganese (II) acetylacetonate, 1,2-hexadecanediol, dodecanoic acid, dodecylamine, benzyl ether, anhydrous dichloromethane, monomethylpolyethylene glycol (mPEG; Mw 1 k, 2 k, 5 k, 10 k, 20 k Da)were purchased from Sigma-Aldrich. All other chemicals and reagents were of analytical grade.
2.2 Synthesis of 6 nm MnFe2O4 Magnetic Nanocrystals
MnFe2O4 nanocrystals were synthesized by seed-mediated growth method [5]. Typically, 2 mmol iron (III) acetylacetonate, 1 mmol manganese (II) acetylacetonate, 10 mmol 1,2-hexadecanediol, 6 mmol dodecanoic acid, 6 m moldodecylamine, and 20 mL of benzyl ether were mixed under a nitrogen atmosphere. The mixture was preheated to 200°C for 120 min and then refluxed at 300°C for 60 min. After being cooled to room temperature, the products were purified with an excess of pure ethanol [12].
2.3 Synthesis of 12 nm MnFe2O4 Magnetic Nanocrystals
2 mmol iron (III) acetylacetonate, 1 mmol manganese (II) acetylacetonate, 10 mmol 1,2-hexadecanediol, 2mmoldodecanoic acid, 2 mmol dodecylamine, and 20 mL of benzyl ether were mixed and magnetically stirred under a flow of N2. Eighty four milligram sample of pre-synthesized 6 nm MnFe2O4 nanoparticles dispersed in hexane (1 mL) was added into the mixture. The mixture was first heated to 110°C for 30 min to remove hexane, then further to 200°C for 1 h. Under a blanket of nitrogen, the mixture was further heated to reflux (300°C) for 30 min. The black-colored mixture was cooled to room temperature by removing the heat source. After being cooled to room temperature, the products were purified with an excess of pure ethanol [12].
2.4 Synthesis of DA-PEG Block Copolymers
DA-PEG block copolymer was synthesized as described previously [6,7].As reported, a solution of 30 mmol dodecanoic acid (DA) and 10 mmol mPEG dissolved in 40 mL of anhydrous dichloromethane was activated by adding 30 mmol of N,N’-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The reaction was carried out for 48 h at room temperature under a nitrogen atmosphere. The resulting product was filtered using a cellulose acetate syringe filter (pore size ≈ 200 nm) and dialyzed for two weeks against 10 mM sodium phosphate buffer (pH 7.4) using dialysis tube.
2.5 Surface coating of MnFe2O4nanocrystals with DA-PEG
30 mg of MnFe2O4nanocrystals was dissolved in 4 mL of chloroform. This organic phase was added to 20 mL of sodium phosphate buffer containing 200 mg of DA-PEG with various sizes of mPEG (1 K, 2 K, 5 K, 10 K and 20 K molecular weight), respectively. After mutual saturation of the organic and continuous phases, the mixture was emulsified for 15 min with an ultrasonicator (ULH700S, Ulssohitech, Korea) at 300 W. After solvent evaporation in 4 h, the product was purified 3 times with centriprep at 3,000 rpm for 30 min to remove excess DA-PEG molecules [9].
2.6 Colloidal Stability
The colloidal stability of the prepared MnMNC-PEGs was determined from their resistance to pH-induced nanoparticle aggregation. A 100 μL nanoparticle suspension (20 mg/mL) was added to 2 mL (pH 2, 4, 74, 9) at room temperature and then size of the suspension was measured using laser scattering (ELS-Z, Otsuka electronics).
2.7 Cell viability assay by MTT
The biocompatibility of the prepared MNC6-PEG1K and MNC12-PEG2K for macrophage cells was quantified by a colorimetric assay based on the mitochondrial oxidation of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). RAW 264.7 cells were harvested at a density of 104 cells/200 μL in a 96-well plate and incubated at 37°C under 5% CO2 atmosphere. The cells were incubated for 24 h with prepared MnMNCs, rinsed with 100 μL PBS (pH 7.4, 1 mM), and then treated with freshly prepared MTT solution (10 μL) and incubated for an additional 4 h before adding 100 μL dimethylsulfoxide. After 24 h, the plates were assayed using an enzyme-linked immunosorbent assay (Spectra Max 340, Molecular Devices, USA) and the results were measured at an absorbance wavelength of 575 nm and a reference wavelength of 650 nm [9].
2.8 MR imaging
We performed MR imaging experiments with a 1.5-T clinical MRI instrument with a micro-47 surface coil (Intera; Philips Medical Systems, Best, the Netherlands). R2 relaxivities of MnMNC6-PEG(1 k ~20 k) and MnMNC12-PEG (1 k ~20 k) were measured using the Carr-Purcell-Meiboom-Gill sequence at room temperature: TR = 10 s, 32 echoes with 12 ms even echo space, number of acquisition = 1, point resolution of 156 × 156 μm, section thickness of 0.6 mm. R2 was defined as 1/T2 with units of s−1. For T2-weighted MR imaging of cells in vitro at 1.5 T, the following parameters were used: point resolution: 156 × 156 μm, section thickness of 0.6 mm, TE = 60 ms, TR = 4000 ms, number of acquisitions = 1. For T2 mapping of cells in vitro, the following parameters were used: point resolution of 156 × 156 μm, section thickness of 0.6 mm, TE = 20, 40, 60, 80, 100, 120, 140, 160 ms, TR = 4000 ms, number of acquisitions =2.
2.9 Prussian blue stain
RAW 264.7 cells (5.0 × 105 cells/well) were seeded onto six-well plates and incubated for 24 h at 37°C. Prepared MnMNC6-PEG1K and MnMNC12-PEG2K (100 μg of MnFe/mL) were added to Dulbecco’s modified eagle medium (DMEM, Gibco®, Invitrogen, USA). After incubation for 24 h at 37°C, the cells with MnMNC6-PEG1K and MnMNC12-PEG2K were detached, centrifuged and washed three times with PBS (pH 7.4, 1 mM). The detached cells were fixed and immersed in iron staining solution (20% hydrochloric acid: potassium ferrocyanate =1: 1) for 30 min at room temperature after being fixed in 95% alcohol for 5 min. Then, the samples were rinsed three times in deionized water to remove the residual staining solution. Subsequently, the samples were stained with the nuclear staining solution (Nuclear Fast Red) for 15 min, followed by three washes with deionized water, and were finally fixed in increasing concentrations of alcohol and xylene [13].
2.10 Characterization
The morphologies and the sizes of the prepared MnMNCs were analyzed using high resolution transmission electron microscopy (HR-TEM, JEM-2100 LAB6, JEOL Ltd., Japan) and laser scattering (ELS-Z, Otsuka electronics, Japan). X-ray diffraction measurement was performed by a Rigaku D/max-RB (Tokyo, Japan) powder diffractometer and image-plate photography using graphite-monochromatized Cu Kα radiation (λ =1.542 Å) to determine the lattice of the MnFe2O4. Data were collected from 20° to 80° with a step size of 0.05° and step time of 5 s. The amounts of metal ions were quantified using inductively coupled plasma atomic emission spectrometry (ICP-AES, Thermo electron corporation, USA).