Morphological effect of oscillating magnetic nanoparticles in killing tumor cells
© Cheng et al.; licensee Springer. 2014
Received: 4 March 2014
Accepted: 17 April 2014
Published: 28 April 2014
Forced oscillation of spherical and rod-shaped iron oxide magnetic nanoparticles (MNPs) via low-power and low-frequency alternating magnetic field (AMF) was firstly used to kill cancer cells in vitro. After being loaded by human cervical cancer cells line (HeLa) and then exposed to a 35-kHz AMF, MNPs mechanically damaged cell membranes and cytoplasm, decreasing the cell viability. It was found that the concentration and morphology of the MNPs significantly influenced the cell-killing efficiency of oscillating MNPs. In this preliminary study, when HeLa cells were pre-incubated with 100 μg/mL rod-shaped MNPs (rMNP, length of 200 ± 50 nm and diameter of 50 to 120 nm) for 20 h, MTT assay proved that the cell viability decreased by 30.9% after being exposed to AMF for 2 h, while the cell viability decreased by 11.7% if spherical MNPs (sMNP, diameter of 200 ± 50 nm) were used for investigation. Furthermore, the morphological effect of MNPs on cell viability was confirmed by trypan blue assay: 39.5% rMNP-loaded cells and 15.1% sMNP-loaded cells were stained after being exposed to AMF for 2 h. It was also interesting to find that killing tumor cells at either higher (500 μg/mL) or lower (20 μg/mL) concentration of MNPs was less efficient than that achieved at 100 μg/mL concentration. In conclusion, the relatively asymmetric morphological rod-shaped MNPs can kill cancer cells more effectively than spherical MNPs when being exposed to AMF by virtue of their mechanical oscillations.
Due to their excellent biocompatibility, monodispersity, and magnetic resonance, iron oxide (Fe3O4) magnetic nanoparticles (MNPs) have been proved useful in various biomedical applications such as contrast agent in magnetic resonance imaging , cellular imaging , drug carrier in targeted drug delivery system [3, 4], and magnetic fluids in hyperthermia [5, 6].
Alternating magnetic field (AMF)-assisted thermal therapy has received widespread attention for tumor treatment recently. In thermal therapy, the localized MNPs in cancerous tissue generate abundant heat via a high-frequency (300 to 1,100 kHz) AMF for an irreversible thermal injury [7, 8]. However, the neighboring healthy tissues may also be injured by the redundant heat. It is proved that the heat generation efficiency of MNPs heavily depends on the particle size and frequency of external AMF [7, 9]. As the particle size increases to micron-sized or AMF frequency decreases, the degree of Néel relaxation and Brownian relaxation decreases, suppressing heat generation. Meantime, AMF-induced vibration or rotation of particles displaces heat generation as the main pattern of AMF energy consumption. In a newly reported research, magnetic microdiscs were used for targeted cancer cell destruction by means of AMF-induced vibrations . In theory, the MNPs reorient in the alternating magnetic field  and the oscillation of immobilized MNPs takes place in situ in the localization of cancerous tissues . Hence, the oscillating MNPs can mechanically damage cancerous tissues at the cellular level as ‘nanoscale scalpel’. It is notable that no thermal damage will be made to the surrounding tissues.
The utilization of forced vibration of MNPs makes the best use of the neglected part of AMF energy consumption. In biomedical applications of forced MNP vibration, patterns and intensity of MNPs' vibration, as well as the degree of thermal damage, will vary according to differences in size, morphology, and exposure concentration of MNPs. By now, most biomedical application research of MNPs related to nanospheres . However, the involvement of rod-shaped MNPs (rMNP) is greater than that of spherical MNPs (sMNP). In this research, an assumption that AMF-induced oscillations of rMNPs can damage cell viability more seriously will be investigated in vitro on human cervical carcinoma cells (HeLa), considering their extensive use in cells uptake and tumor therapy research [14–16]. Similarly sized rod-shaped (length 200 ± 50 nm, diameter 50 to 120 nm) and spherical (diameter 200 ± 50 nm) Fe3O4 MNPs in three different concentrations were synthesized and used to investigate the effects of MNP morphology and concentration in killing tumor cells.
Synthesis of MNPs
Spherical Fe 3 O 4 MNPs FeCl3 · 6H2O (0.81 g) was dissolved in 25 mL glycol and transferred to a 50-mL teflon-lined stainless steel autoclave. KAc (1.47 g) was then added to the solution, stirring constantly. Autoclave was sealed and maintained at 200°C for 24 h. After naturally cooled to room temperature, the black magnetite particles were gathered by magnet and washed with deionized water and ethanol three times, respectively. The final product was dried in a vacuum at 60°C for 12 h.
Rod-shaped Fe 3 O 4 MNPs Rod-shaped MNPs were synthesized following the procedure described previously . Stoichiometric FeSO4 · 7H2O (0.139 g), FeCl3 · 6H2O (0.270 g), and 5 mL ethylenediamine were sealed in the autoclave and maintained at 120°C for 12 h. After naturally cooled to room temperature, the rMNPs were gathered by magnet and then post-processed with the same steps for sMNPs.
The prepared MNPs were ultrasonically treated to break up clusters and then sterilized using 75% (v/v) ethanol. The sterile MNPs were dissolved in DMEM medium at concentrations of 20, 100, and 500 μg/mL.
Material characterization: TEM, XRD, and VSM
Morphology and size of MNPs were observed by transmission electron microscopy (TEM) (H-800; Hitachi, Chiyoda, Tokyo, Japan) operating at 200 kV. Composition and crystal form were characterized by X-ray diffraction (XRD) (D/MAX 2200; Rigaku, Tokyo, Japan) with Cu Kα radiation (λ = 0.154056 nm), with operation voltage at 40 kV and current at 40 mA. Magnetic properties including the saturation magnetic induction and coercivity were measured by vibrating sample magnetometer (VSM) (Lakeshore 7407; Lake Shore Cryotronics Inc., Westerville, OH, USA).
Quantification of MNPs' loading
HeLa cells (Cell Bank at the Chinese Academy of Science, Shanghai, China) were seeded at a density of 104 cells/well in a 96-well plate. After 2 h incubation at 37°C in 5% CO2 atmosphere, the cells were exposed to the culture medium containing MNPs at concentrations of 20 (low), 100 (medium), or 500 μg/mL (high) for 3, 6, 12, or 20 h. At four desired time points, cells were rinsed with phosphate-buffered saline (PBS) to remove unfixed MNPs. Then, the MNP-loaded cells in each well were fully dissolved by hydrochloric acid (37.5%, w/v). At last, ferrozine solution (10 mg/mL) was added, and the absorbance of complex of ferrozine and ferrous ion was measured using spectrophotometer (UV 3100; Shanghai Mapada Intruments Co., Ltd., Shanghai, China). Ferrous ions were quantified by referencing the corresponding standard curve.
Treatment of MNP-loaded HeLa cells
HeLa cells were cultured in 50 mL tissue culture flask at 37°C in 5% CO2 atmosphere with three concentrations of MNPs as stated above, and the optimized incubation time was selected based on the quantification results. After incubation, the cells were rinsed with PBS twice to remove the unfixed MNPs. Then, the dissociated cells were equally divided into five 0.5-mL centrifuge tubes and centrifuged at 1,000 rpm for 3 min. Finally, the cell pellet was placed in the center of AMF with culture medium covering the cells and then treated by the AMF device for 0, 10, 30, 60, or 120 min, respectively. AMF treatments of MNPs and MNP-loaded cells were performed at 37°C in airtight conditions. The temperature of cell pellet was recorded by the infrared thermometer (OS 3708; Omega Engineering, Stamford, CT, USA).
Cell viability assay: MTT assay and trypan blue assay
MTT assay Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK) assay. After being treated in AMF, HeLa cells were reseeded into 96-well petriplate for 2 h incubation in quintuplicate. Following incubation, 20 μL MTT (5 mg/mL in PBS) solution was added to each well and incubated for another 4 h. After that, the culture supernatant was extracted, and purple insoluble MTT product was re-dissolved in 150 μL dimethyl sulfoxide. Lastly, the concentration of the reduced MTT in each well was measured at 570 nm using a microplate reader. It is notable that the untreated MNP-loaded cells (i.e., the 0 min group) were used as control and absorbance was adjusted by correcting for the bias caused by the dark MNPs.
Trypan blue assay After being treated with AMF, the medium was removed and the cells were stained by 0.4% trypan blue (Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK) solution for 3 min. The cells with damaged cell membranes were stained by trypan blue and counted under the optical microscope. The above tests were repeated three times.
Optical images of cellular semi-thin sections, SEM of cell surface, and TEM of cellular ultramicrocuts
The HeLa cells were firstly fixed by adding 0.5% and 2% (w/v) glutaraldehyde and kept for 1 h at room temperature. Then the cells were dehydrated with ethanol in series of concentrations 50%, 70%, 80%, 90%, and 100% (v/v) for 10 min respectively. Finally, the acetone-infiltrated cells were embedded in resin, and the blocks containing the cells were cut into thin sections in 500 or 50 nm using a diamond knife. For TEM of internal cell structure, the 50-nm ultramicrocuts were transferred into a copper grid for viewing. For optical macroscope viewing (6XB-PC, Shanghai Optical Instrument Factory, Shanghai, China), the 500-nm semi-thin sections were observed. For scanning electron microscope (SEM; LEO1530VP; LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) of cell surfaces, the dehydrated cells were conductively coated and observed at 5 kV.
Results and discussion
Thermal effect of AMF
During the AMF treatment, neither type of MNPs leads to an obvious temperature rise. This is because of the low power and low frequency of the device relative to the commonly used thermal therapy device [18, 19]. When 0.1 g solid MNPs powder was placed in the center of the AMF-generating device, the maximal temperature rise was 1.7°C. It is known that the required temperature for irreparable cell damage during hyperthermia therapy should be no less than 43°C [20, 21]. Additionally, the relative small mass fraction of MNPs was used in the treated unit. Therefore, this marginal temperature rise suggested that the thermal injury could be neglected in this study.
Cell cytotoxicity and characterization of cell loading
Cell viability after AMF treatment
Mechanisms of morphological effect
In this research, AMF-induced oscillation of MNPs was proved able to mechanically damage cancer cells in vitro, especially when relatively asymmetric rod-shaped MNPs were used. Additionally, the concentration of MNPs affects the efficiency of AMF treatment. In this study, AMF treatment was most efficient when cells were in advance culture in medium containing MNPs at a concentration of 100 μg/mL and treated for 2 h or more.
This work was supported in part by The National Nature Science Foundation of China (10805069, 10875163) and Shanghai Pujiang Programme (13PJ1401400).
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