Mn–Fe Nanowires Towards Cell Labeling and Magnetic Resonance Imaging

2.1. Synthesis of the nanostructures

Spontaneous organization of small individual nanostructures into large and well-defined nanowires, represents (Tang et al., 2002; Grubbs 2007) a facile way to obtain useful nanomaterials for magnetic devices (Hangarter et al., 2007; Wu et al., 2007). The key to prepare our target 1-D Mn-Fe nanowires for cell labeling and MRI contrast could be achieved in such a way that small building blocks — MnFe₂O₄ nanoparticles which were prepared by co-precipitation at elevated temperature (Sousa et al., 2001; Aquino et al., 2002; Deng et al., 2005), were rationally organized into Mn-Fe nanowires with larger lengths in relatively high yields. We have found out that suitable amount of linker — cystamine (NH₂CH₂CH₂S–SCH₂CH₂NH₂) could induce the organization of the as-synthesized MnFe₂O₄ nanoparticles into novel amine-functionalized Mn-Fe nanowires in good yields under basic condition and magnetic stirring for 24 hours. The precipitate was separated by centrifugation and washed with water/ethanol mixture to afford the 1-D Mn-Fe nanostructures.

2.2. Characterization of the nanostructures

The morphology of as-prepared MnFe₂O₄ nanoparticles was characterized by high-resolution TEM, revealing their morphologies with an average diameter of 5 nm (Figure 1A). One drop of sample in ethanol suspension was added to the holey carbon-coated

copper grid and was allowed to evaporate to dryness. For the prepared Mn-Fe nanostructures, TEM images revealed that they were in substantial amounts (Figures 1B-D). The nanoneedles possessed (Figure 1B) an average length of 400 nm and width of 8 nm; while the nanorods possessed (Figure 1C) an average length of 800 nm and width of 30 nm. For the nanowires, they possessed (Figure 1D) an average length of 1 μm and width of 35 nm.

In addition to the TEM characterization, inductively coupled plasma-optical emission spectroscopy (ICP-OES) and energy-dispersive X-ray (EDX) spectroscopy were employed for the determination of elemental contents (Mn, Fe, S, O and N) of the nanoarchitectures (Table 1, Figure 2). EDX measurements were performed by locating a region (~20 nm × 20 nm) with substantial amount of materials on copper grid without carbon coating. By comparing between the EDX spectra of the MnFe₂O₄ nanoparticles (Figure 2A) and nanowires (Figure 2B), the spectrum of the nanowires revealed additional signals of sulfur and nitrogen as well as enhanced signals of carbon, originating from the attached cystamine linker. The observed EDX signal of copper, which originates from the TEM sample grid, has been omitted in the calculations of the elemental contents present in our nanostructures. The X-ray diffraction (XRD) analysis revealed that the MnFe₂O₄ nanoparticles exhibited several peaks corresponding to the characteristic interplanar spacings 220, 311, 400, 511 and 400 of the spinel structure with 2θ 31.5, 35.0, 42.4, 56.2 and 61.7, respectively. These results are similar to the reported values in the literature (Aquino et al., 2002).

ICP-OES samples were dissolved in 2% HCl solution with a few drops of SnCl₂ solution. Iron absorption was observed at 238.20 nm while the manganese absorption was observed at 257.61 nm. Although the separate measurements (Table 1) by ICP-OES and EDX occurred with the errors that are less than 1%, there existed relatively large errors (0.3–48%) when comparing the results obtained in both methods. Generally, ICP-OES is regarded as an accurate method to determine the metal ion concentrations while EDX spectroscopy is an evaluation of the existence of elements in a specific area on sample grid surface. The ratio of Mn:Fe is approximately 1:2. From the MnFe₂O₄ nanoparticles to the nanowires, there was a trend in the quantitative ICP-OES measurements that both the Mn and Fe metal contents decrease slightly. Moreover, the sulfur and nitrogen contents originated from the cystamine linker appeared in the EDX measurement of the 1-D nanoarchitectures. These increased organic characteristics indicate that the amounts of linker play a crucial role in controlling the sizes of the nanostructures.

The magnetic properties of the nanomaterials have been investigated using a vibrating sample magnetometer (VSM). The VSM spectra (M-H) of magnetization (emu/g) versus magnetic field (Oe) are examined. Figure 3A shows a hysteresis loop of typical MnFe₂O₄ nanoparticles measured by sweeping the external field between –10000 to 10000 Oe at room temperature. The magnetization curve shows no remanemce or coercivity at room temperature, demonstrating the superparamagnetic character. Superparamagnetism is the responsiveness to an applied magnetic field without retaining any magnetism after removal of the applied magnetic field. The saturated magnetization value of MnFe₂O₄ nanoparticles is determined to be 43.7 emu/g. On the other hand, Figure 3B shows a hysteresis loop of Mn-Fe nanowires measured by sweeping the external field between –10000 to 10000 Oe at room temperature. The magnetization curve shows that the coercivity and remanence are small at room temperature (Figure 3B inset). The saturated magnetization value of Mn-Fe nanowires is determined to be 9.0 emu/g. The decreased saturated magnetization value is in part due to the increasing thickness of organic coating layer of the nanowires. Other reasons are the presence of a magnetic dead or anti-ferromagnetic organic layer on the nanowire’s periphery.

For high-density information storage, the superparamagnetic relaxation of magnetization direction in tiny magnetic data bits has to be avoided in order to keep the digital data stored for a desired period of time. For biomedical applications such as magnetic resonance imaging, hyperthermia, drug delivery, and catalysis, in contrast, superparamagnetic property of materials would be essential from which the materials do not retain any magnetization in the absence of an externally applied magnetic field. In our system, soft magnetized nanowires have been employed as contrast agents for magnetic resonance imaging.

2.3. Magnetic resonance imaging

For magnetic resonance imaging (MRI), the capability of nanoarchitectures to influence the T2 relaxation time was studied using a clinical 1.5 T whole-body MR system (Sonata, Siemens Symphony, Erlangen, Germany) with a standard human knee coil radio frequency coil for excitation and signal reception. Nanoarchitectures were diluted in distilled water at nanowire concentrations of 100, 10, 1 and 0.1 µg/mL. For MR measurements, the nanomaterial suspensions (1 mL) were filled in 2 mL Eppendorf vials each. These vials were placed in a water bath. Sonication was applied for 10 min prior to MRI using at 35 kHz in water at ambient temperature. The imaging sequence was a standard Carr-Purcell-Meiboom-Gill pulse sequence with the following parameters: TR = 2000 milisecond, TE range 30-960 milisecond, 32 echoes, FOV = 134 × 67 mm², matrix = 128 × 64, slice thickness 5 mm, NEX = 3. T2 relaxation times were calculated by a linear fit of the logarithmic ROI signal amplitudes versus TE.

When a cylindrical magnetic nanorod segment is disk-shaped, its magnetization axis lies perpendicular to the rod axis. It also follows that if the segment is longer than its width, its axis of magnetization is parallel to the nanorod axis (Ferré et al., 1997). Therefore, the magnetic properties of nanostructures can be tuned over a wide range by tuning the aspect ratio of the magnetic block and its composition (Fert & Piraux 1999). Experimentally, the T2 relaxivities (r2) for nanoneedles, nanorods, and nanowires were determined to be 20.81 ± 0.58, 8.10 ± 0.31, and 6.62 ± 0.42 mM^-1sec^-1, respectively (Figure 4). These values are at the lower end of the relaxivity of other iron oxide nanoparticle derivatives, such as VSOP-C184 has a r2 of 33 mM^-1sec^-1 and SHU-555C (Supravist) has a r2 of 38 mM^-1sec^-1 (Wang et al., 2001; Nitin et al., 2004; Lee et al., 2007; Lu et al., 2007; Lee et al., 2008). It is interesting to notice that though the iron concentration of nanoneedles is not very different to the other nanostructures, it has significantly higher MR relaxivity than the other nanostructures. It is known that MR relaxivity depends in part on the crystal lattice of the iron oxide. Our results suggest that the nanoneedles with higher MR relaxivity are majorly composed of iron oxide crystal lattice similar to those of the nanoparticles (Wang et al., 2001). The development of larger 1-D nanostructures leaded to lower MR relaxivities. Despite the slightly weaker MR relaxivity of the nanostructures developed in the current study, it is expected that the unique shape of these nanostructures can find applications for cell labeling, as drug carrier, magnetic separation of labeled cells and other biological entities, and also in the application of magnetic targeting (Alexiou et al., 2000; Pankhurst et al., 2003; Tartaj et al., 2003; Berry & Curtis 2003). It may be possible to further modify the systems with the aim to increase their relaxivities. It has been reported that the doping of Mn into Fe₃O₄ can also increase the MR

relaxivity as compared with the pure iron oxide counterpart (Lee et al., 2007). For in vivo tissue contrast, the weaker MR relaxivity can be compensated by using T2* MRI sequence and higher magnetic field such as 3 Tesla or 7 Tesla.

The nanowires were incubated with RAW264.7 cells followed by Prussian blue staining. The results indicated that the Mn-Fe nanowires were effectively incorporated into RAW264.7 cells without the addition of any transfecting agent (e.g., liposomes) (Figure 5). Figures 5B–5D reveal the optical microscopic images of the RAW264.7 cells incubated with nanowires for 2 hours at the concentration of 10, 50, 100 µg/mL respectively. As increasing

the labeling concentration with nanowires, increased amounts of the nanowire uptake were observed as in the blue-stained part inside the cells. At the nanowire concentrations of 50 and 100 µg/mL (Figures 5C and 5D), the cell labeling efficacies with the nanowires were approximately 100%.

TEM analysis was also performed to confirm the location of nanowires inside the RAW264.7 cells. For the TEM analysis, specimens were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.05% CaCl₂ for 2 hours at room temperature, followed by post-fixation in 2% osmium tetroxide in the same buffer solution for 2 hours at room temperature. After dehydration and embedded in Spurr’s resin, ultra-thin sections (80 nm) were cut before examined under a FEI/Philips Tecnai 12 TEM operated at 80 kV. TEM results demonstrated that the nanowires were located within the lyzosome and cell vesicles (Figure 6). No obvious sub-cellular superstructure injury and cell apoptosis was observed. However, modest amount of the nanoparticles were observed inside the lyzosome and cell vesicles, a result which indicates that the nanostructures might be susceptible to enzymatic degradation in the slightly acidic lyzosome environment. It is also expected that some nanowires incorporated in the cells were fragmented or partially cut during the preparation of the ultra-thin section for TEM.

It has been demonstrated that certain biocompatible synthetic materials with amine functional peripheries could enhance the degree of cell adhesion and transfection (Pollard et al., 1998; Wang et al., 2009). As a result, our reported Mn-Fe nanostructures which contain free amines would be beneficial not only to the macrophage cells but also can extend their labeling efficacy to other cell lines.

To assess the biocompatibility of the nanostructures, after RAW264.7 cells incubated with 0.1, 1, 10, 50, 100 µg/mL nanowires for 2 hours, Trypan blue exclusion assay (Sigma T6146) was performed to assess the viability of the cells. To assess the cell proliferation potential post labeling, RAW264.7 cells were cultured in 96-well plate at the density of 2500 cell/well with DMEM including 10% FCS. After labeling cells with 0.1, 1, 10, 50 or 100 µg/mL nanowires for 2 hours, nanowires were removed from the plate and PBS was used to rinse the residual nanostructures. Fresh DMEM including 10% FCS was added again for normal cell growth. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to detect the proliferation of the nanowire-labeled RAW264.7 cells.

The Trypan blue exclusion assay results and MTT assay results are shown in Figure 7. RAW264.7 cell viability was not significantly affected from the labeling concentration of 0.1 up to 50 µg/mL with nanowires, and no apparent growth inhibition of RAW264.7 cells was observed after labeling up to 50 µg/mL of nanowires (inclusive). These results revealed the satisfactory safety profiles of these nanostructures.