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2008, 112, 15085–15088 Published on Web 09/10/2008
Induction of Cell Death by Magnetic Actuation of Nickel Nanowires Internalized by Fibroblasts Andrew O. Fung,† Vishal Kapadia,‡ Erik Pierstorff,‡,§ Dean Ho,*,‡,§,| and Yong Chen*,⊥ Biomedical Engineering IDP, UniVersity of California at Los Angeles, Box 951600, 5121 Engineering 5, Los Angeles, California 90095-1600, Department of Biomedical Engineering, Department of Mechanical Engineering, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208, Robert H. Lurie ComprehensiVe Cancer Center, Northwestern UniVersity, 303 E. Superior Street, Chicago, Illinois 60611, Department of Mechanical and Aerospace Engineering, UniVersity of California at Los Angeles, Box 951597, 38-137G Engineering 4, Los Angeles, California 90095-1597 ReceiVed: July 13, 2008; ReVised Manuscript ReceiVed: August 7, 2008
Magnetic nanomaterials with multimodal functionalities have emerged as a versatile platform for biomedical applications that range from basic cellular interrogation to clinical nanomedicine. In this work, we have prepared electrodeposited ferromagnetic nickel nanowires for efficient internalization into 3T3 fibroblasts. Agitation of the nanowires by a low external field induced cell death, as assessed by MTT viability assays. The response of the interleukin-6 (IL-6) gene expression of the fibroblasts to nanowire-mediated cellular manipulation was examined by quantitative real-time polymerase chain reaction (qRT-PCR). These nanowires exhibited significant potential as therapeutic and interrogative platforms for biomedicine. Introduction The properties of engineered magnetic nanoparticles lend to several attractive applications in biomedicine. Their highly controllable size range promotes intimate contact with cells and subcellular structures. They can be functionalized with biomolecules to facilitate their penetration into tissue for contrast enhancement in imaging or localized drug delivery. Furthermore, their magnetic properties allow for actuation by an external field gradient. The negligible magnetic response of biological tissues to magnetic fields facilitates the integration of nanoparticles and biology for noninvasive cellular interrogation. Biomedical applications have been well reviewed by Pankhurst et al.1 and include selection of rare cells in blood,2 monitoring of live-cell cytoskeletal remodeling,3 and treatment of cancers by hyperthermia.4 While the majority of biomedical studies have used spherical para/magnetic nanoparticles, relatively few applications have leveraged the favorable properties of ferromagnetic nanowires. Spherical particles respond to magnetic flux gradients, whereas the high aspect ratio of nanowires allows the application of torque with relatively weak external fields. It was recently shown that nickel (Ni) nanowires could be internalized by neuroblasts and then used to transport the cells to ferromagnetic micropatterns.5 In this paper we demonstrate the induction of fibroblast cell death by actuation of internalized Ni nanowires, with a view toward dynamic targeted cell therapy. Real-time PCR (RT-PCR) quantification of interleukin-6 (IL* To whom correspondence should be addressed. E-mail: yongchen@ seas.ucla.edu;
[email protected]. † Biomedical Engineering IDP, University of California at Los Angeles. ‡ Department of Biomedical Engineering, Northwestern University. § Department of Mechanical Engineering, Northwestern University. | Robert H. Lurie Comprehensive Cancer Center, Northwestern University. ⊥ Department of Mechanical and Aerospace Engineering, University of California at Los Angeles.
10.1021/jp806187r CCC: $40.75
6) is used to investigate the cellular response to the nanowires. The impact of a weak rotating magnetic field on cell viability is measured by MTT assay. Experimental Methods Ni nanowires were fabricated by electrodeposition into porous aluminum oxide templates.5,6 A 60 µm-thick aluminum oxide membrane with 0.2 µm nominal pore diameter (Whatman, Anodisc) was sputter coated on one side with approximately 20 nm of gold. The membrane was sealed with the gold-coated side to a copper sheet. Ni was potentiostatically electrodeposited into the pores at -1.5 V dc relative to a Ni wire (Alfa Aesar, #41361) for 20 min from a solution of 1.14 M NiSO4, 0.19 M NiCl2, and 0.73 M H3BO3 (Sigma-Aldrich). The gold was then removed with a small amount of gold etchant (Transene, TFA) at 60 °C, and the aluminum oxide template was removed by immersion in 6 M NaOH. The resulting mixture was centrifuged for 30 s at 50 rcf to precipitate relatively large particles of the template material and any Ni that electrodeposited outside the pores. The supernatant containing the nanowires was rinsed four times by centrifugation for 3 min at 200 rcf followed by resuspension in nanopure water and then suspended in Dulbecco’s Modified Eagle’s Medium (ATCC) with 10% v/v bovine-calf serum (DMEM-BCS). Separately, labeled nanowires for fluorescent imaging were prepared by incubation in DMEM-BCS with 10 µg/ml Alexafluor635-streptavidin conjugate (Invitrogen) with agitation for 1 h at room temperature. These labeled nanowires were centrifuged and resuspended in fresh culture medium to a final concentration of approximately 0.25 mg/ml. NIH/3T3 fibroblasts (ATCC, CRL-1658) were cultured in DMEM-BCS at 37 °C and 5% CO2 according to the vendor protocol to approximately 90% confluence. Nanowires in 2008 American Chemical Society
15086 J. Phys. Chem. C, Vol. 112, No. 39, 2008
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Figure 1. Electrodeposition of Ni nanowires in Anodisc membranes. (a) Pore structure of unfilled alumina membrane; scale bar 200 nm. (b) Cross-section of alumina membrane; scale bar 200 nm. (c) Ni nanowires after release from the alumina membrane; scale bar 1 µm.
Green I (Molecular Probes), and fluorescein (Bio-Rad), using oligo-dT cDNA as the PCR template. For cell viability studies, 3T3 fibroblasts were cultured in a 96-well plate (BD Falcon). The plate was then put on a hotplate stirrer (VWR, 12365-382) at 37 °C for 20 min with a magnet of 240 mT field rotating at 1 Hz. Cell cultures were further incubated for 20 min in the culture chamber before their viability was measured by mitochondrial dehydrogenase activity (MTT assay) according to the vendor protocol (Sigma-Aldrich, TOX1). Samples were prepared in triplicate and optical absorbance was measured using a plate reader (Tecan, Safire). Figure 2. Confocal projection and cross-sections of 3T3 fibroblast (red) with several internalized Ni nanowires (blue). FM 4-64 membrane stain clearly shows nanowires in the intracellular space. Scale bar 10 µm.
suspension were added to the cells in culture and incubated for approximately 12 h. For imaging, the cells were stained with 5 µg/ml FM 4-64 membrane stain (Invitrogen) in Hank’s Buffered Salt Solution (HBSS), fixed with formalin solution (Sigma-Aldrich) for 10 min at 25 °C, and washed in three changes of phosphate buffered saline (PBS). Imaging was performed with the Leica SP2 confocal microscope. For qRT-PCR analysis of the expression of IL-6, 3T3 fibroblasts were cultured in 6-well plates (BD Falcon). Total RNA was isolated from the cell cultures with TRIZOL (Invitrogen) according to manufacturer’s protocol. cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s protocol. PCR was then performed using the iCycler thermocycler (Bio-Rad). qRT-PCR was conducted in a final volume of 25 µL containing Taq polymerase, 1X Taq buffer (Stratagene), 125 µM dNTPs, SYBR
Results and Discussion The diameter of the nanowires is determined by the template pore diameter, and the length is controlled by the deposition time. The pore diameter of the unfilled alumina membrane was observed by SEM to be in the range of 198–280 nm (Figure 1). The 20 min deposition yielded nanowires with an average length of 4.4 µm (3.0-6.0 µm mean, standard deviation 820 nm). Thus, a nanowire of the average length and nominal diameter had an aspect ratio of 22. The upper limit of torque exerted by the external magnetic field on a single nanowire can be estimated with consideration of its magnetic properties. The magnetic properties of these nanowires are dominated by shape anisotropy due to their large aspect ratio and the low magnetocrystalline anisotropy of Ni. The magnetic easy axis of an isolated nanowire lies on its long axis.7 The external magnetic field, b B, exerts aligning torque, b σ, on the magnetic moment, m b , of the nanowire according to the relationship
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J. Phys. Chem. C, Vol. 112, No. 39, 2008 15087
b b τ)m b × B ) µ0m b×H
(1)
b is the magnetic field strength, µ0 is the permeability Where H b, of free space, and m b is the product of the magnetization, M and volume, V, of the nanowire
b ·V m b)M
(2)
Similar Ni nanowires that are described in the literature b s, values in the range of have saturation magnetization, M 2 -1 8,9 46-50 A m kg . This value is significantly below the accepted room-temperature value for bulk Ni (55.4 A m2 kg-1). This has been attributed to the presence of a nickel oxide layer on the surface of the nanowires. Let us assume b s, nanowire ) 0.9M b s, bulk. Using remnant magnetization, that M Mr, the squareness ratio is defined as Mr/Ms. According to the dependence of the squareness ratio on the aspect ratio reported by Das et al.,10 we estimate the maximum Mr/Ms ) 0.26. Thus the nanowire of average dimensions would experience an estimated maximum torque of 1.3 × 10-14 N m in this experimental setup if it were previously magnetically saturated. The nanowires, however, were not magnetized to saturation in the present work to avoid agglomeration and the need to disperse the nanowires by sonication. Thus, this estimate can be taken as an upper limit of the actual torque. Remarkably, the estimated forces exerted by the nanowires on the cells are much smaller than even the stall force for the elongation of a single Actin filament (approximately 2 pN).11 Cross-sectional reconstructions of the confocal microscopy data confirmed that the fibroblasts internalized the nanowires
(Figure 2). This is consistent with previous work that used fibroblast, osteoblast, and neuroblast cells.8 The cells maintained normal morphology and adherence after 12 h exposure to nanowires (Figure 3a). Ni in particulate and ionic forms has great potential to interact with and alter the intracellular environment.12 It has been reported to be a potent agent of hypersensitivity,13 and is moderately cytotoxic.14 IL-6 is a pro-inflammatory cytokine that is significantly upregulated in fibroblasts in response to local tissue trauma. Gene expression of IL-6 was measured to examine the inflammatory response of cells exposed to the nanowires (Figure 3a). There was little to no inflammation after 12 h exposure to the nanowires (Figure 3b). The absence of IL-6 upregulation following cellular incubation with the nanowires suggests their potential use as biocompatible therapeutic vehicles. Given the known effects of Ni on the upregulation of IL-6, the apparent lack of cytotoxicity observed here may be explained by the presence of oxide layers that form by reaction with NaOH during the removal of the template.8 Serum proteins that adsorb to this oxide may further buffer the Ni from proinflammatory interactions with the intracellular environment. The MTT assay showed that cell viability was not significantly affected by the nanowires in the absence of an applied field (Figure 3c). Application of a rotating magnetic field for 20 min decreased the viability of fibroblast cultures by 89% (Figure 3d,e). The molecular crowding of cytoplasmic macromolecules and cytoskeletal filaments15 hinders the movement of internalized nanowires. Thus, a force exerted by the nanowires would immediately couple to cytoplasmic structures. To the best
Figure 3. Inflammatory response and viability of fibroblasts exposed to Ni nanowires; scale bars 20 µm. (a) Fibroblast culture after 12 h exposure to Ni nanowires. (b) IL-6 expression was not significantly affected after 12 h exposure to Ni nanowires. (c) MTT assay of culture showing no significant change in viability due to exposure to Ni nanowires. (d) Fibroblast culture from panel a following 20 min exposure to rotating magnet of 240 mT field. (e) MTT assay of culture in panel d showing an 89% reduction in viability.
15088 J. Phys. Chem. C, Vol. 112, No. 39, 2008 of our knowledge, this is the first instance of magnetically actuated cell death by disruption of the intracellular environment with nanowires. The precise mechanism of apoptosis or necrosis caused by this mechanical interference of the cell structure is the subject of further study. This magnetic induction of cell death offers an alternative to hyperthermia for local cellular catabolism. Magnetic particle hyperthermia16 uses a time-varying magnetic field and the hysteresis loss of nanoparticles to transfer toxic levels of thermal energy to the adjacent tissue. One of the challenges in the effective biomedical application of hyperthermia is the ability to deliver sufficient nanoparticles to produce adequate heating of the target tissue. The task is further constrained by the clinical acceptability of ac magnetic field conditions,17 which limits the product of the magnetic field strength and frequency H · f < 4.85 × 108 A m-1 s-1. While the majority of nanoparticles used in hyperthermia studies are approximately spherical, the work described here exploits the magnetic anisotropy of high-aspect-ratio nanowires and cellular internalization to achieve significant cell death with lowfrequency weak magnetic fields. The nanowire action can be finely controlled by the frequency, strength, and duration of the applied magnetic field. Moreover, the biochemical functionalization of the nanowires can be engineered for mediated endocytosis.18 The combination of cell targeting and dynamic delivery raises potential applications in actuated cell-specific therapy. Conclusion In this work we have developed electrodeposited ferromagnetic Ni nanowires for the interrogation and manipulation of NIH/3T3 fibroblasts. The internalization of the nanowires alone, verified by confocal microscopy, did not cause upregulation of IL-6. The nanowires were subsequently actuated by a weak, low-frequency magnetic field to effectively induce cell death, which was confirmed by MTT assays. While previous studies have used nanoparticle-driven hyperthermia to induce catabolism, this work exploited the magnetic anisotropy of high-aspectratio nanowires to demonstrate their utility as therapeutic agents. The subsequent engineering of additional functionality into the ferromagnetic nanowires (e.g., conjugation of drug payload, cell-
Letters specific targeting, etc.) may lead to significantly enhanced multimodal therapy and diagnostics for nanoscale medicine. Acknowledgment. The authors thank Dr. I. D. Goldberg for very helpful discussions about magnetic properties of nanowires. A.F. gratefully acknowledges a NSERC postgraduate scholarship for study abroad. D.H. gratefully acknowledges support from a V Foundation for Cancer Research Scholar Award. This work was also supported by the Center on Functional Engineered and Nano Architectonics (FENA) at UCLA, and NSF Center for Scalable and Integrated NanoManufacturing (SINAM). References and Notes (1) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167. (2) Liberti, P. A.; Rao, C. G.; Terstappen, L. J. Magn. Magn. Mater. 2001, 225, 301. (3) Reed, J.; Troke, J. J.; Schmit, J.; Han, S.; Teitell, M. A.; Gimzewski, J. K. ACS Nano 2008, 2, 841. (4) Moroz, P.; Jones, S. K.; Gray, B. N. Int. J. Hyperthermia 2002, 18, 267. (5) Choi, D.; Fung, A.; Moon, H.; Ho, D.; Chen, Y.; Kan, E.; Rheem, Y.; Yoo, B.; Myung, N. Biomed. MicrodeVices 2007, 9, 143. (6) Bentley, A. K.; Farhoud, M.; Ellis, A. B.; Lisensky, G. C.; Nickel, A. M. L.; Crone, W. C. J. Chem. Educ. 2005, 82, 765. (7) Xu, X.; Zangari, G. J. Appl. Phys. 2005, 97. (8) Prina-Mello, A.; Diao, Z.; Coey, J. J. Nanobiotechnol. 2006, 4, 9. (9) Hultgren, A.; Tanase, M.; Felton, E. J.; Bhadriraju, K.; Salem, A. K.; Chen, C. S.; Reich, D. H. Biotechnol. Prog. 2005, 21, 509. (10) Das, B.; Mandal, K.; Sen, P.; Bandopadhyay, S. K. J. Appl. Phys. 2008, 103. (11) Footer, M. J.; Kerssemakers, J. W. J.; Theriot, J. A.; Dogterom, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2181. (12) Kasprzak, K. S.; Sunderman, F. W.; Salnikow, K. Mutat. Res. 2003, 533, 67. (13) Garner, L. A. Dermatol. Ther. 2004, 17, 321. (14) Schmalz, G.; Schuster, U.; Schweikl, H. Biomaterials 1998, 19, 1689. (15) Seksek, O.; Biwersi, J.; Verkman, A. S. J. Cell Biol. 1997, 138, 131. (16) Bae, S.; Lee, S. W.; Takemura, Y.; Yamashita, E.; Kunisaki, J.; Zurn, S.; Kim, C. S. IEEE Trans. Magn. 2006, 42, 3566. (17) Atkinson, W. J.; Brezovich, I. A.; Chakraborty, D. P. IEEE Trans. Biomed. Eng. 1984, 31, 70. (18) Wuang, S. C.; Neoh, K. G.; Kang, E. T.; Pack, D. W.; Leckband, D. E. Biomaterials 2008, 29, 2270.
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