MAGNETIC NANOPARTICLE LABELING
OF CULTURED CANCER CELL LINE WITHOUT
TRANSFECTION AGENT
Jong-Kai Hsiao
∗,†, Chung-Yi Yang
∗,†, Yiao-Hong Wang
†,‡,
Chen-Wen Lu
†, Borade Prajakta Uttam
†, Hon-Man Liu
†and Jaw-Lin Wang
∗,§ ∗Institute of Biomedical Engineering
National Taiwan University, Taipei, Taiwan
†
Department of Medical Imaging
National Taiwan University Hospital and College of Medicine
Taipei, Taiwan
‡
Institute of Electrical-Engineering
National Taiwan University
Taipei, Taiwan
Accepted 26 May 2008
ABSTRACT
Magnetic nanoparticle (MNP) labeling of stem cell has been proved its efficacy for cell trafficking. Most of the labeling technique requires mixture of iron oxide nanoparticles and transfection agent. Stem cells with ionic MNP without the aid of transfection agent were labeled previously. The possibility of high efficiency labeling of cultured cancer cell, HeLa cell, by using ionic MNP is proposed. The labeled cell morphology was observed and the intracellular iron content was determined by spectrophotometry. The cell character change was evaluated by flow cytometry where front scattering count and side scattering count (SSC) were recorded. The imaging ability of the labeling method was determined by T2 weighted magnetic resonance (MR) imaging. Labeled MNPs were accumulated at cytoplasm is observed and the iron content of labeled cell could reach 27 pg/cell. There is no cell diameter change but the cell granularity increased according to SSC data from flow cytometry. Under clinical 1.5 T MR imaging, we could detect labeled cells easily were detected at the cell number of 1×105. It is concluded that labeling of cancer cell with ionic MNPs without transfection agent is an efficient labeling method which will provide non-invasive imaging method for monitoring cancer behavior.
Keywords: Magnetic nanoparticle; Magnetic resonance imaging; HeLa cell; Iron oxide.
INTRODUCTION
Magnetic nanoparticles (MNPs) composed of iron oxide are widely used as magnetic resonance imaging con-trast medium.1 The cores of these nanoparticles are
made up of either Fe3O4 or r-Fe2O3.1,2 To prevent particle aggregation or precipitation, the particles are coated with either dextran or carboxydextran.3,4 Both iron oxide and dextran derivatives are biocompatible
§Corresponding author: Jaw-Lin Wang, Institute of Biomedical Engineering, National Taiwan University. No. 1, Sec. 1,
Ren-ai Rd., Taipei, Taiwan. Tel.: +886-2-33665269; Fax: +886-2-33665268; E-mail: jlwang@ntu.edu.tw
added.8,9There is a debate on the influence of transfec-tion agent on the labeled cells.10 Most of the concerns are alteration of differentiation ability of the stem cells. We thus developed a labeling technique by using ionic MNPs instead of non-ionic MNPs. The efficiency is high enough that we can detect labeled stem cells in a single cells level under clinical 1.5 Tesla MRI.11
HeLa cells are cervical cancer cell line that has been used widely for investigating cancer biology.12It is rec-ognized by its immortal behavior owing to the telom-erase activity. Labeling of HeLa cells with ionic MNPs would help us understand its behavior once if the HeLa cells have been implanted in the living organism. More-over, it would further prove the cell labeling capacity by using ionic MNPs. Consequently, we investigated the feasibility of labeling HeLa cells with ionic MNPs by analyzing labeling efficacy, cell viability, cell morphol-ogy, and Magnetic Resonance (MR) imaging.
MATERIAL AND METHODS
Cell Culture and Iron Oxide
Treatment
HeLa cell was purchased from the Culture Collec-tion and Research Center (CCRC, Hsin-Chu, Tai-wan). The cells were cultured in Dulbecco’s modified Eagle Medium (DMEM) (Cellgro, Herndon, VA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (0.05 mg/ml). The cells were incubated at 37◦C in 5% CO2. For incubation with MNPs, Ferucarbotran, a clin-ically approved ionic MNPs was added to the culture medium at concentrations of 1, 10 or 100µg Fe/ml respectively which corresponds to 0.11, 1.1, and 11 times plasma concentration after intravenous adminis-tration of Ferucarbotran at suggested dosage.13
Iron Content Measurement
Iron concentration of HeLa cell was determined as described in Refs. 13 and 14. Briefly, 1×105 SPIO
The cells were imaged using an inverted microscope (Nikon Eclipse TS100, Tokyo, Japan) at 200× magnifi-cation. Prussian blue staining was used for localization of the intracellular iron oxide as described in Ref. 15. Flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA) was applied for the determination of granularity change and cell diameter between MNPs treated and non-treated groups.16Briefly, macrophages were collected by measuring side scattering counts (SSC) and front scattering counts (FSC). For each group of macrophages, 10,000 counts were measured and the results were graphed with the assistance of com-puter software (CellQuest, Becton Dickinson, San Jose, CA, USA).
MRI Image
The confirmation of MNPs loading was further inves-tigated by MR imaging as described in Ref. 17. HeLa cells were treated with 0, 1, 10 or 100µg Fe/ml of MNPs for 1, 4, and 18 h and then harvested, centrifuged in a 200µl test tube. Up to 1 × 105 cells were collected for MR imaging. These cell sam-ples were positioned in the water bath, and then placed into 1.5 Tesla clinical MRI system equipped with a eight channel head colid (Signa Excite, GE Healthcare NJ, USA) and scanned under T2 weighted gradient echo pulse sequences (TR = 550, TE = 5, FA = 15◦, FOV = 16 cm× 8 cm, resolution = 256 × 192, slice thickness = 1.4 mm, spacing = 0.3 mm, NEX = 3). The acquired images were analyzed by the image work-station provided by the vendor.
RESULTS
Prussian blue stain of HeLa cells revealed abundant deep blue granules in the cytoplasm of cells treated with 100µg Fe/ml MNPs and could also be found at lesser degree in cells exposed to MNPs at the concentration of 10µg Fe/ml. The cell nucleus is not stained. The blue staining could not be found in cells exposed to MNPs at the concentration of 0.1µg Fe/ml (Fig. 1).
(A) (C)
(B) (D)
Fig. 1 Microscopic view of HeLa cells labeled with ionic MNPs at 0 µg Fe/ml (A), 1 µg Fe/ml (B), 10 µg Fe/ml (C) and 100 µg Fe/ml (D) for 18 h. The cells were stained with Prussian blue and the images were taken at the magnification of 20×.
For quantification of MNPs uptake into cells, HeLa cells exposed to 1, 10, and 100µg Fe/ml of MNPs were further analyzed for their iron content. We observed that the time and dose dependent manner of iron content increase. The iron content inside the cell is richest in cells exposed to 100µg Fe/ml of MNPs for 18 h. The iron content reached 27 pg/cell, which is 1.9 times higher compared to cells treated for only 1 h (Fig. 2).
The cell behavior changes were determined by flow cytometry. The FSC versus SSC was measured and the distribution indicated the cell morphology and the cell debris. There is no significant increase in cell debris in each groups compared with control group where no ionic MNPs were added (Fig. 3).
The pattern of histograms of FSC was measured as a means for evaluating cell morphology. There is no evidence of cell size change in all of the groups which was evidenced by similar count peak at the same level (Fig. 4).
The pattern of SSC was shifted to left side when the cells were treated with higher concentration of ionic MNPs and longer exposure time (Fig. 5(A)). The pattern could be demonstrated more clearly at merged
Iron content of labeled HeLa cells
0.000 5.000 10.000 15.000 20.000 25.000 30.000 1 10 100
Nanoparticle concentration ( g Fe/mL)
pg/Cell
1 hour 4 hours 18 hours
Fig. 2 Photospectrometric analysis of iron content of HeLa cells labeled with different concentration of ionic MNPs with different incubation time. The concentration is 1, 10, and 100µg Fe/ml and the incubation time varied from 1, 4 to 18 h.
Fig. 3 Flow cytometry analysis of ionic MNPs labeled HeLa cells at 1, 4, and 18 h with different MNPs concentration from 0, 1, 10 to 100µg Fe/ml. The scattered plots represented the cell distribution at different size and granularity. The cell debris and dead cells are represented as small clusters of dots at left lower corner of each plot.
Fig. 4 Cell diameter measurement by histograms of FSC of ionic MNPs labeled HeLa cells for 18 h. The red arrows indicate the peak number of cell diameter. The cells were exposed to ionic MNPs at 0, 1, 10, and 100µg Fe/ml, respectively.
plot where HeLa cells were exposed to MNPs for 18 h at different MNPs concentration (Fig. 5(B)).
The cells were further investigated for its label-ing ability by directly scanned under 1.5 T clinical MRI. There is marked signal intensity drop in cells treated with 100µg Fe/ml MNPs at 18 h. The sig-nal intensity drop also revealed incubation dose and time-dependent manner. There is no signal intensity change at control group, whereas the signal intensity drop could be observed in cells treated from 10 to 100µg Fe/ml when the incubation time reaches 18 h (Fig. 6).
DISCUSSION
Labeling of cells with MNPs for cell trafficking provide a non-invasive method for monitoring cell implantation.
However, transfection agent such as protamine sulfate is usually needed for facilitating cell labeling. We previ-ously reported a new method for labeling human mes-enchymal stem cells without transfecting agent. Using this technique we can observe labeled cell at single cell level.11We proved in this paper that the same labeling method could be applied into cancer cells. Besides, the labeling efficiency is comparable in HeLa cell which has 27 pg Fe/cell compared to human mesenchymal stem cells that has 23.4 pg Fe/cell in our previous study. This finding further support the application ability of this labeling method.
We reported that the ingested MNPs will be trans-ported to membrane bound intracellular organelle which was supposed to be lysosomes.18 This phe-nomenon also supports the finding of SSC increase in cells with MNPs uptake. This measuring method would
(A)
(B)
Fig. 5 (A) Cell granularity measured by histograms of SSC at different concentrations of ionic MNPs and exposure time interval. The concentration was 0, 1, 10, and 100µg Fe/ml, respectively, whereas the exposure time was 1, 4, and 18 h. (B) Merged histogram revealed cell granularity change at different ionic MNPs concentration. These cells were exposed to MNPs for 18 h.
Fig. 6 Cross-sectional T2 weighted MR image of test tubes filled with HeLa cells labeling with ionic MNPs at different con-centration and different exposure duration. The concon-centration was 0, 1, 10, and 100µg Fe/ml and the MNPs exposure time is 1, 4, and 18 h.
be a fast, robust technique to analyze MNPs labeling efficiency since the MNPs attached to the cell mem-brane will not be detected.
We previously noted that the iron content of the macrophage cell was correlated with labeling efficiency
proved by flow cytometry and MRI signal intensity change.18The same correlation could be found in cancer cells. Labeling cancer cells with MNPs have potential benefits in the study of cancer biology. First, it pro-vides us a non-invasive method for monitoring cancer invasion. Second, at the assistance of high resolution MRI, single cancer cell could be detected and be moni-tored for cancer cell migration, chemotaxis, and metas-tasis. Third, it is known that cancer cells will interact with adjacent cells such as macrophages.19And we just developed T1 contrast nanorods.20 By labeling these two kinds of cells with either T1 or T2 contrast particle, the interaction between these cells could be observed in living organism, which work could only partially be done by intravital microscopy.
Although we proved the labeling efficiency of ionic MNPs in cancer cells, the uptake mechanism, and its potential effect toward cancer cells is not fully understand. The MNPs uptake pathway was proposed to be clathrin receptor related, consequently, future studies by different uptake inhibitor assay should be analyzed.17
cell has different cell behavior including rapid prolifera-tion, migraprolifera-tion, and invasion compared to stem cells.22 Understanding these effects might have benefits in the treatment of malignancy.
CONCLUSION
We have developed high efficiency labeling method and imaging ability of HeLa cell, one kind of cancer cell by using ionic MNPs without transfecting agent. The opti-mum labeling efficiency was incubation at the concen-tration of 100µg Fe/ml for 18 hours. This method will provide us the opportunity for studying cancer biology in living animal by clinical MR imaging facility.
REFERENCES
1. Moghimi SM, Hunter AC, Murray JC, Nanomedicine: Current status and future prospects, FASEB J 19(3):311–330, 2005.
2. Oude Engberink RD, van der Pol SMA, Dopp EA, de Vries HE, Blezer ELA, Comparison of SPIO and USPIO for in vitro labeling of human monocytes: MR detection and cell function, Radiology243(2):467–474, 2007. 3. Chouly C, Pouliquen D, Lucet I, Jeune JJ, Jallet P,
Development of superparamagnetic nanoparticles for MRI: Effect of particle size, charge and surface nature on biodistribution, J Microencaps 13(3):245–255,
1996.
4. Reimer P, Balzer T, Ferucarbotran (Resovist): A new clinically approved RES-specific contrast agent for contrast-enhanced MRI of the liver: Properties, clinical development, and applications, Euro. Radiol.
13(6):1266–1276, 2003.
5. Arbab AS, Liu W, Frank JA, Cellular magnetic res-onance imaging: Current status and future prospects,
Expert Rev Med Dev3(4):427–439, 2006.
6. Bremer C, Allkemper T, Baermig J, Reimer P, RES-specific imaging of the liver and spleen with iron oxide particles designed for blood pool MR-angiography,
J Mag Res Imaging10(3):461–467, 1999.
7. Kraitchman DL, Heldman AW, Atalar E, Amado LC, Martin BJ, Pittenger MF, Hare JM, Bulte JWM, In vivo magnetic resonance imaging of mesenchy-mal stem cells in myocardial infarction, Circulation
107(18):2290–2293, 2003.
lowes V, Read EJ, Frank JA, Labeling of cells with ferumoxides–protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells, NMR Biomed18(8):553–559, 2005.
11. Hsiao JK, Tai MF, Chu HH, Chen ST, Li H, Lai DM, Hsieh ST, Wang JL, Liu HM, Magnetic nanoparticle labeling of mesenchymal stem cells without transfec-tion agent: Cellular behavior and capability of detectransfec-tion with clinical 1.5 T magnetic resonance at the single cell level, Mag Res Med58(4):717–724, 2007.
12. Lai CW, Wang YH, Lai CH, Yang MJ, Chen CY, Chou PT, Chan CS, Chi Y, Chen YC, Hsiao JK, Iridium-complex-functionalized Fe3O4/SiO2 core/shell nanoparticles: A facile three-in-one system in magnetic resonance imaging, luminescence imaging, and photo-dynamic therapy, Small4(2):218–224, 2008.
13. Metz S, Bonaterra G, Rudelius M, Settles M, Rummeny EJ, Daldrup-Link HE, Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro, Euro Radiol14(10):1851–1858, 2004.
14. Matuszewski L, Persigehl T, Wall A, Schwindt W, Tombach B, Fobker M, Poremba C, Ebert W, Heindel W, Bremer C, Cell tagging with clinically approved iron oxides: Feasibility and effect of lipofection, particle size, and surface coating on labeling efficiency,
Radiol-ogy235(1):155–161, 2005.
15. Arbab AS, Yocum GT, Kalish H, Jordan EK, Ander-son SA, Khakoo AY, Read EJ, Frank JA, Efficient mag-netic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI, Blood104(4):1217–1223, 2004.
16. Hsiao J-K, Tai M-F, Yang C-Y, Chen S-T, Wang J-L, Ku H-C, Liu H-M, Comparison of micrometer and nanometer sized magnetic particles for cell labeling,
Magnetics, IEEE Trans43(6):2421–2423, 2007.
17. Lu CW, Hung Y, Hsiao JK, Yao M, Chung TH, Lin YS, Wu SH, Hsu SC, Liu HM, Mou CY, Yang CS, Huang DM, Chen YC, Bifunctional magnetic silica nanoparti-cles for highly efficient human stem cell labeling, Nano
Lett7(1):149–154, 2007.
18. Hsiao JK, Tai MF, Lee YC, Yang CY, Wang HY, Liu HM, Fang JS, Chen ST, Labeling of cultured macrophages with novel magnetic nanoparticles, J Mag
Mag Mater304(1):e4–e6, 2006.
19. Chen JJW, Lin YC, Yao PL, Yuan A, Chen HY, Shun CT, Tsai MF, Chen CH, Yang PC, Tumor-associated macrophages: The double-edged sword in cancer pro-gression, J Clin Oncol23(5):953–964, 2005.
20. Tsai CP, Hung Y, Chou YH, Huang DM, Hsiao JK, Chang C, Chen YC, Mou CY, High-contrast para-magnetic fluorescent mesoporous silica nanorods as a multifunctional cell-imaging probe, Small4(2):186–191, 2008.
21. Magnitsky S, Watson DJ, Walton RM, Pickup S, Bulte JWM, Wolfe JH, Poptani H, In vivo and ex vivo MRI
detection of localized and disseminated neural stem cell grafts in the mouse brain, Neuroimage 26(3):744–754, 2005.
22. Hsiao J-K, Law B, Weissleder R, Tung C-H, In-vivo imaging of tumor associated urokinase-type plasmino-gen activator activity, J Biomed Opt 11(3):034013– 034015, 2006.
