Cell Creeping and Controlled Migration by Magnetic Carbon Nanotubes
© to the authors 2009
Received: 28 March 2009
Accepted: 5 October 2009
Published: 27 October 2009
Carbon nanotubes (CNTs) are tubular nanostructures that exhibit magnetic properties due to the metal catalyst impurities entrapped at their extremities during fabrication. When mammalian cells are cultured in a CNT-containing medium, the nanotubes interact with the cells, as a result of which, on exposure to a magnetic field, they are able to move cells towards the magnetic source. In the present paper, we report on a model that describes the dynamics of this mammalian cell movement in a magnetic field consequent on CNT attachment. The model is based on Bell’s theory of unbinding dynamics of receptor-ligand bonds modified and validated by experimental data of the movement dynamics of mammalian cells cultured with nanotubes and exposed to a magnetic field, generated by a permanent magnet, in the vicinity of the cell culture wells. We demonstrate that when the applied magnetic force is below a critical value (about F c ≈ 10−11 N), the cell ‘creeps’ very slowly on the culture dish at a very low velocity (10–20 nm/s) but becomes detached from the substrate when this critical magnetic force is exceeded and then move towards the magnetic source.
Carbon nanotubes (CNTs)  are molecular-scale tubes of graphite carbon with unique properties including extreme strength, electric properties and other characteristics , which account for their large scientific and industrial interest with thousands of original publications on nanotubes being reported every year. CNTs are either single-wall CNTs (SWCNTs) consisting of a single graphite lattice rolled into a perfect cylinder or multi-wall CNTs (MWCNTs) made up of several concentric cylindrical graphite shells. One characteristic that accounts for their scientific and clinical relevance to the biomedical field is the ability of CNTs to penetrate plasma membranes. This property has driven research and development, which has resulted in significant advances in CNT chemistry and functionalization specifically for the use of CNTs as vectors for the delivery of a spectrum of therapeutic substances, e.g., peptides, proteins, nucleic acids and drugs, to cells and tissues . More recent reported biological studies have been based on work, which has exploited their unique physical properties. These include the strong near infrared absorbance by SWCNTs for tumour cell ablation , bacterial electroporation by field emission properties of MWCNTs  and localized heat release from SWCNTs following application of a radiofrequency radiation also for thermal ablation of cancer . CNTs also possess intriguing magnetic properties, which derive from the metal catalyst impurities entrapped at CNT extremities during their manufacture, enabling them to react to external magnetic fields. This property has been utilized by Cai et al. to develop an alternative physical method of nanotube ‘spearing’ for in vitro and in vivo gene transfection of cells with plasmid DNA . Monch et al. have shown that ferromagnetic filled carbon nanotubes can interact with human bladder cancer EJ28 cells . Based on this magnetic property, we have recently demonstrated that MWCNTs when exposed to a magnetic field are able to interact with cells and induce their migration towards the magnetic source . Controlled migration of mammalian cells could have important potential clinical applications, e.g., in cancer therapy to curtail the metastatic behaviour of invasive cancer , accelerating regeneration after peripheral nerve injuries, etc. In the present paper, we report on the measurement and modelling of CNT-induced cell movement. Mammalian cells cultured with MWCNTs were tracked and studied for 3 days in order to document and measure their migration dynamics. These data were then used to develop and validate a model, which describes this CNT-induced cell movement in a magnetic field.
Materials and Methods
Human SH-SY5Y cells (ATCC CRL-2266) were grown using a mixture (1:1) of Ham’s F12 and DMEM supplemented with 2 mMl-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated fetal bovine serum (FBS). Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air/5% CO2. CNT-modified medium was obtained by adding PF-127 coated MWCNTs to the cell culture medium at a ratio 1:10 (v/v). Spectrometric and FIB analyses performed over 5 weeks from sample preparation revealed a great stability of the solution (no phenomena of nanotube aggregation or precipitation). Lipofectamine (GenePORTER 2; Genlantis, San Diego, CA) was used for the transfection of a plasmid DNA containing a green fluorescent protein (gfp) gene reporter in SH-SY5Y cells according to the protocol provided by the supplier. After transfection, cells were cultured without any experimental manipulation for 24 hours before any further experimental testing. The transfection efficiency was about 80% (percentage of GFP-positive cells counted by fluorescent microscopy). Optical and fluorescent microscopy was performed with a Nikon TE2000U fluorescent microscope equipped with Nikon DS-5MC USB2 cooled CCD camera. To determine the effect of carbon nanotubes and the surfactant on cell viability, we used WST-1 (tetrazolium salt 2-(4-iodophenyl)-3-(4-nitophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoilium) cell proliferation assay. Then, 25 × 10³ cells were seeded into each well of a 96-well plate and then incubated with the culture media for 72 h. The culture medium was then replaced with 100 μL of medium containing 10 μL of WST-1 solution (as described in quick cell proliferation assay kit, BioVision, USA) and incubated for 2 h in standard conditions. The absorbance was measured on a Versamax microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 450 nm with background subtracted at 650 nm.
Cell Migration Assays
Values are reported as mean ± standard error of the mean (S.E.M.). The WST-1 experiments were carried out in triplicate. One-way statistical analysis of variance (ANOVA) followed by post hoc comparison test (Turkey test) was performed; a P value <0.001 was considered significant.
Results and Discussion
Cell Migration on Exposure to a Magnet Field
where r is the distance from the magnet.
We have observed experimentally that such a magnetic force induces cell migration. The adhesion of a cell to a surface is mediated by reversible bonds between specific receptor-ligand molecules . The magnetic force operates by overcoming the receptor-ligand bonds, which are responsible for the very slow movement (quasi-stationary) state of the cell.
where E 0 is the free energy change on binding, r s is the binding cleft, f is the force applied per bond and kT = 4.1 × 10−21 J is the thermal energy. In the literature, for a representative antigen–antibody bond, E 0 is estimated about 5.9 × 10−20 J, the binding cleft r s = 0.5 nm within a factor of 2 and τ0 in the order of 10−8 s .
The model results plotted in Fig. 8 were achieved with r s = 1 nm (corresponding to one bond broken for each elementary displacement) and τ0 = 5 × 10−8 s. The overlap between the model and experimental data is quite good (R 2 = 0.967), and the parameters are in the range of values referred to above.
Carbon nanotubes produced by CCVD of hydrocarbon sources on substrates impregnated with metal catalysts entrap at their extremities metal particles, which confer them magnetic properties. We have previously reported that such nanotubes when added to the cell culture medium induce cell migration towards a permanent magnet. This paper proposes a model of cell movement based on the theory of bond survival postulated by Bell in 1978. The model parameters, i.e., nanotubes magnetic susceptibility and the amount of CNTs entrapped per cell were estimated experimentally. In vitro tests of cell migration dynamics confirmed goodness of fit with the proposed model. The model predicts that SH-SY5Y cells cultured with 10 μg/mL of nanotubes ‘creep’ on the culture disk (at a velocity about 10 nm/s) until the applied magnetic force reaches a critical value (F c ≈ 10−11N), which causes cell detachment and migration towards the magnetic source. In view of the potential clinical application, further studies are needed to study the in vivo behaviour and function of mammalian cells tagged with CNTs and subjected to the effect of an external magnetic field.
The authors would like to thank Prof. Hiroyuki Nishide, Prof. Shinji Takeoka, Mr. Takeshi Ibe and Mr. Yosuke Obata from the School of Advanced Science and Engineering, Waseda University, Tokyo for their kind support during the SQUID analysis
The activity presented in this work has been partially supported by the NINIVE (Non Invasive Nanotransducer for In vivo gene therapy, STRP 033378) project, co-financed by the 6FP of the European Commission and by the IIT (Italian Institute of Technology) Network. Authors gratefully thank Mr. Carlo Filippeschi for his kind support using the FIB microscope.
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