A systematic study of core size and coating thickness on manganese-doped nanocrystals for high T2 relaxivity as magnetic resonance contrast agent
© Park et al.; licensee Springer. 2015
Received: 29 July 2014
Accepted: 20 September 2014
Published: 3 February 2015
We describe a systematic study of coating thickness and their effect on different core sizes for the optimized preparation of highly sensitive manganese-doped magnetic nanocrystals (MnMNCs) to be served as magnetic resonance (MR) contrast agent. From these efforts, MnMNCs with 12 nm core and DA-PEG2k coating demonstrated that T2 relaxivity (r2) was increased by 7.29-fold (r2 value: 452 mM−1 s−1) compare to conventional iron oxide (CLIO) and remarkable colloidal stability in various physiological conditions. Further in vitro cellular MR imaging results showed that MnMNC-PEGs were biocompatible and well suited for medical applications. This study will provide a useful synthetic strategy for the development of highly effective MR contrast agents.
Magnetic nanocrystals (MNCs) are emerging research areas for their potential applications in biomedical sciences such as magnetic resonance imaging, cancer treatment and micro-NMR sensors [1–3]. Researchers are aiming to make high sensitive magnetic materials by means of increasing size, engineering magnetism, and surface coating variations [4–6]. Manganese-doped magnetic nanocrystals (MnMNCs) are one of the most important material because of relatively simple preparation method and increased mass magnetization value compare to conventional iron oxides . However MnMNCs are coated with hydrophobic ligands and eventually they need to be encapsulated in hydrophilic ligands to disperse them in water phase. Our group reported a method to make highly water dispersible MnMNCs, they are still in suboptimal potency of sensitivity because magnetic field surrounding a magnetic nanocrystals would be fallen as the distance go far from the core material. And the coating molecules are important factor that would affect the nuclear relaxation of water protons by forming hydrogen bond. Therefore, it is necessary work to study the affect of coating materials even if their iron oxide core sizes are similar [8–11].
Herein, we report on an optimized design for manganese-doped magnetic nanocrystals (MnMNCs), capable of achieving maximal r2 and colloidal stability (Figure 1). These synthesized MnMNCs with different coating thickness can exhibit quite different r2 even if their MnMNC core sizes remain similar. These constructions could approach the maximum r2 coefficient for a given material. As a specific example in the present study, we synthesized MnMNCs for T2-weighted magnetic resonance (MR) imaging contrast agent and encapsulated into a various PEG shell. The resultant MnMNCs with 12 nm core size encapsulated with dodecanoic acid-PEG2K Da (MnMNC12-PEG2K) exhibited most high r2 relaxivity (452 mM−1 s−1 [metal]), 7.29-fold higher compared to conventional iron oxide (CLIO), and showed excellent colloidal stability. The prepared MnMNC12-PEG2K was subsequently applied to an intra-cellular imaging system to demonstrate their use in identifying target cells in vitro.
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 . 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 .
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 .
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 .
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 .
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 .
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).
3 Results and discussion
Calculated [ 14 ] and measured thickness of different core and PEG sizes
PEG 1 K (N = 22)
PEG 2 K (N = 45)
PEG 5 K (N = 113)
PEG 10 K (N = 226)
PEG 20 K (N = 454)
10.8 ± 1.7
12.8 ± 1.4
18.8 ± 2.7
22.8 ± 2.1
26.8 ± 1.5
21.6 ± 2.8
23.6 ± 3.2
28.6 ± 2.7
34.6 ± 2.4
41.6 ± 2.6
In summary, we developed an efficient coating method on different core size MnFe2O4 magnetic nanocrystals (MnMNCs) using dodecanoic acid (DA)-PEG amphiphilic block copolymers by solvent evaporation method, and determined r2 value maximized MnMNCs based MR probes as MRI contrast agents compared to conventional iron oxide nanoparticles (Feridex and CLIO). In addition, DA-PEG coating of MnMNCs was stable and offered an optimal shell/core to achieve successful colloidal stability as well as low cytotoxicity. The study on the combination of optimal PEG layer thickness and magnetic core size for producing maximal r2 coefficient revealed that MnMNCs with 12 nm core coated with DA-PEG2K (MnMNC12-PEG2K) provided the highest r2 coefficient for MR imaging. Consequently, these advantageous features of optimized PEG coated MnMNCs allowed us to obtain outstanding MR imaging results demonstrating the utility of this MR contrast agent design in future diagnostic MR imaging applications.
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012050077).
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