In the past two decades, our understanding in biology, materials science and nanotechnology has expanded rapidly. The inevitable intersection of these three disciplines has set in motion the development of an emerging research area, nanobiotechnology or nanobiomedical science, which offers exciting and abundant opportunities for discovering new processes and phenomena. In particular, the advances in the synthesis and characterization of nanoscale materials allow scientists to understand and control the interactions between nanomaterials (e.g., nanowires, nanofibers, nanoparticles, nanobelts or nanoribbons, and nanotubes) and biological entities (e.g., nucleic acid, proteins, or cells) at molecular or cellular levels. These advances promise major achievements in the life sciences.
By way of an example, the research on magnetic nanomaterials (Skomski, 2003) has attracted a lot of attention because of their numerous applications including magnetic separation of biomolecules (Nam et al., 2003), as biocompatible contrast agents for magnetic resonance imaging (MRI) (Wang et al., 2001; Berry & Curtis 2003; Pankhurst et al., 2003; Tartaj et al., 2003; Nitin et al., 2004; Lee et al., 2007), magnetic recording (Darques et al., 2009), spintronic devices and magnetic sensing (Thurn-Albrecht et al., 2000; Allwood et al., 2002; Redl et al., 2003). Relatively large, near-monodisperse spherical iron oxide (Fe3O4, magnetite) nanoparticles with average core diameters of 200–1000 nm are commercially available (e.g., Feridex), some of them are used as contrast agents for
The properties of one-dimensional (1-D) nanowires toward biological systems are attracting increasing attention recently (Fang & Kelley 2009; Cohen-Karni et al., 2009). Generally, nanomaterials having elongated shapes and correspondingly increased surface area are more effective
Although different magnetic nanowires have been fabricated by solution methods—a bottom-up approach (versus a top-down aprroach which produces nanostructures by refinement of bulk materials) (Xia et al., 1999; Stephanopoulos et al., 2005)—and characterized by various techniques (Whitney et al., 1993; Meier et al., 1996; Doudin et al., 1996; Ferré et al., 1997; Fert & Piraux 1999; García & Miltat 2002; Nielsch et al., 2002; Chen et al.(a), 2003; Chen et al.(b), 2003; Love et al., 2003; Ponhan & Maensiri 2009), however, the effective preparation, cytotoxicity, as well as cell labelling efficacy of different cell types using relatively rigid, long magnetic nanowires have been seldomly investigated. Therefore, we have recently investigated rigid 1-D magnetic nanostructures as effective contrast agents for MRI, and discovered the preparation of Mn-Fe oxide nanowires with amine-functional peripheries, which are formed by a self-assembling organization of their corresponding small MnFe2O4 nanoparticles — a process of assembling approach using cystamine as the linker. This approach, which utilizes chemicals and supramolecular driving forces to arrange small components into an ordered conformation, represents an effective and inexpensive way to achieve more complex and functional nanoarchitectures. Herein, the properties of Mn-Fe nanowires with lengths ranging from 400-1000 nm and widths ranging from 8-35 nm for MRI contrast and the potential of macrophage cell uptake are also reported (Leung et al., 2008; Leung et al., 2009).
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 (
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
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
relaxivity as compared with the pure iron oxide counterpart (Lee et al., 2007). For
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.
In summary, the manganese-doped iron oxide nanoparticles are capable to organize into one-dimensional magnetic Mn-Fe nanostructures of different sizes with cystamine. The use of these materials which are employed as the cell-labeling agent for magnetic resonance imaging (MRI), has been explored. All nanoarchitectures demonstrate remarkable magnetic resonance
Alexiou C. Arnold W. Klein R. J. Parak F. G. Hulin P. Bergemann C. Erhardt W. Wagenpfeil S. Lübbe A. S. 2000 Locoregional cancer treatment with magnetic drug targeting.
Allwood D. A. Xiong G. Cooke M. D. Faulkner C. C. Atkinson D. Vernier N. Cowburn R. P. 2003Submicrometer ferromagnetic NOT gate and shift register.
Aquino R. Tourinho F. A. Itri R. Lara M. C. F. L. Depeyrot J. 2002 Size control of MnFe2O4 nanoparticles in electric double layered magnetic fluid synthesis.
Berry C. C. Curtis A. S. G. 2003 Functionalisation of magnetic nanoparticles for applications in biomedicine.
Chen M. Searson P. C. Chien C. L. 2003 Micromagnetic behavior of electrodeposited Ni/Cu multilayer nanowires.
Chen M. Sun L. Bonevich J. E. Reich D. H. Chien C. L. Searson P. C. 2003 Tuning the response of magnetic suspensions.
Cohen-Karni T. Timko B. P. Weiss L. E. Lieber C. M. 2009 Flexible electrical recording from cells using nanowire transistor arrays.
Corot C. Robert P. Idée J. M. Port M. 2006 Recent advances in iron oxide nanocrystal technology for medical Imaging.
Darques M. Spiegel J. De la Torre Medina. J. Huynen I. Piraux L. 2009Ferromagnetic nanowire-loaded membranes for microwave electronics.
Deng H. Li X. Peng Q. Wang X. Chen J. Li Y. 2005Monodispersed magnetic single-crystal ferrite microspheres.
Doudin B. Blondel A. Ansermet J.-Ph 1996 Arrays of multilayered nanowires.
Du L. Chen J. Qi Y. Li D. Yuan C. Lin M. C. Yew D. T. Kung H. F. Yu J. C. Lai L. 2007 Preparation and biomedical application of a non-polymer coated superparamagnetic nanoparticle.
Fang Z. Kelley S. O. 2009 Direct electrocatalytic mRNA detection using PNA-nanowire sensors.
Ferré R. Ounadjela K. George J. M. Piraux L. Dubois S. 1997 Magnetization processes in nickel and cobalt electrodeposited nanowires.
Fert A. Piraux L. 1999 Magnetic nanowires.
Fortin J. P. Wilhelm C. Servais J. Ménager C. Bacri J. C. Gazeau F. 2007 Size-sorted anionic iron oxide nanomagnets as colloidal mediators for magnetic hyperthermia.
García J. M. Miltat A. T. J. 2002 MFM imaging of nanowires and elongated patterned elements.
Grubbs R. B. 2007Solvent-tuned structures.
Hangarter C. M. Rheem Y. Yoo B. Yang E. H. Myung N. V. 2007 Hierarchical magnetic assembly of nanowires.
Lee J. H. Huh Y. M. Jun Y. W. Seo J. W. Jang J. T. Song H. T. Kim S. Cho E. J. Yoon H. G. Suh J. S. Cheon J. 2007 Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging.
Lee H. Y. Lee S. H. Xu C. Xie J. Lee J. H. Wu B. Koh A. L. Wang X. Sinclair R. Wang S. X. Nishimura D. G. Biswal S. Sun S. Cho S. H. Chen X. 2008 Synthesis and characterization of PVP-coated large core iron oxide nanoparticles as an MRI contrast agent.
Leung K. C. F. Wang Y. X. Wang H. H. Chak C. P. 2008Novel one-dimenstional Mn-Fe oxide nanowires for cell labeling and magnetic resonance imaging.
Leung K. C. F. Wang Y. X. J. Wang H. H. Xuan S. Chak C. P. Cheng C. H. K. 2009 Biological and magnetic contrast evaluation of shape-selective Mn-Fe nanowires.
Love J. C. Urbach A. R. Prentiss M. G. Whitesides G. M. 2003 Three-dimensional self-assembly of metallic rods with submicron diameters using magnetic interactions.
Lu C. W. Hung Y. Hsiao J. K. Yao M. Chung T. H. Lin Y. S. Wu S. H. Hsu S. C. Liu H. M. Mou C. Y. Yang C. S. Huang D. M. Chen Y. C. 2007 Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labelling.
Lübbe A. S. Alexiou C. Bergemann C. 2001 Clinical applications of magnetic drug targeting.
Meier J. Doudin B. Ansermet J.-Ph 1996 Magnetic properties of nanosized wires.
Mitragotri S. Lahann J. 2009 Physical approaches to biomaterial design.
Nam J. M. Thaxton C. S. Mirkin C. A. 2003 Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins.
Nielsch K. Hertel R. Wehrspohn R. B. Barthel J. Kirschner J. Gösele U. Fischer S. F. Kronmüller H. 2002Switching behavior of single nanowires inside dense nickel nanowire arrays.
Nitin N. La Conte L. E. W. Zurkiya O. Hu X. Bao G. 2004 Functionalization and peptide-based delivery of magnetic nanoparticles as an intracellular MRI contrast agent.
Pankhurst Q. A. Connolly J. Jones S. K. Dobson J. 2003 Applications of magnetic nanoparticles in biomedicine.
Park J. H. von Maltzahn G. Zhang L. Derfus A. M. Simberg D. Harris T. J. Ruoslahti E. Bhatia S. N. Sailor M. J. 2009 Systematic surface engineering of magnetic nanoworms for in vivo tumor targeting.
Pollard H. Remy J. S. Loussouarn G. Demolombe S. Behr J. P. Escande D. 1998 Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.
Ponhan W. Maensiri S. 2009 Fabrication and magnetic properties of electrospun copper ferrite (CuFe2O4) nanofibers.
Redl F. X. Cho K. S. Murray C. B. O’Brien S. 2003 Three-dimensional binary superlattices of magnetic nanocrystals and semiconductor quantum dots.
Skomski R. 2003Nanomagnetics.
Sousa M. H. Tourinho F. A. Depeyrot J. da Silva. G. J. Lara M. C. F. L. 2001 New electric double-layered magnetic fluids based on copper, nickel, and zinc ferrite nanostructures.
Stephanopoulos N. Solis E. O. P. Stephanopoulos G. 2005 Nanoscale process systems engineering: toward molecular factories, synthetic cells, and adaptive devices.
Tang Z. Kotov N. A. Giersig M. 2002 Spontaneous organization of single CdTe nanoparticles into luminescent nanowires.
Tartaj P. del Puerto Morales. M. Veintemillas-Verdaguer S. Teresita-Carreño González. T. Serna C. J. 2003 The preparation of magnetic nanoparticles for applications in biomedicine.
Thurn-Albrecht T. Schotter J. . Kastle G. A. . Em Ley. N. . Shibauchi T. . Krusin-Elbaum L. Guarini K. Black C. T. Tuominen M. T. Russell T. P. 2000 Ultrahigh-density nanowire arrays grown in self-assembled diblock copolymer templates.
Wang H. H. Wang Y. X. J. Leung K. C. F. Au D. W. T. Xuan S. Chak C. P. Lee S. K. M. Sheng H. Zhang G. Qin L. Griffith J. F. Ahuja A. T. 2009 Durable mesenchymal stem cell labelling using polyhedral superparamagnetic iron oxide nanoparticles.
Wang Y. X. J. Hussain S. M. Krestin G. P. 2001 Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging.
Whitney T. M. Jiang J. S. Searson P. C. Chien C. L. 1993 Fabrication and magnetic properties of arrays of metallic nanowires.
Wilhelm C. Gazeau F. 2008 Universal cell labelling with anionic magnetic nanoparticles.
Wu H. Zhang R. Liu X. Lin D. Pan W. 2007 Electrospinning of Fe, Co, and Ni nanofibers: synthesis, assembly, and magnetic properties.
Xia Y. Rogers J. A. Paul K. E. Whitesides G. M. 1999 Unconventional methods for fabricating and patterning nanostructures.