Inorganic phosphate nanorods are a novel fluorescent label in cell biology
© Patra et al; licensee BioMed Central Ltd. 2006
Received: 28 July 2006
Accepted: 30 October 2006
Published: 30 October 2006
We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4·H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 μg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes
Nanotechnology, the creation of new objects in nanoscale dimensions, is a cutting edge technology having important applications in modern biomedical research [1–7]. Because the dimension of nanoscale devices is similar to cellular components such as DNA and proteins [8, 9], tools developed through nanotechnology may be utilized to detect or monitor several diseases at the molecular level [3, 10, 11]. Bio-imaging with inorganic fluorescent nanorods probes have recently attracted widespread interest in biology and medicine [1–4, 12–14] compared to nanospheres. According to the reported literature , there is a drastic reduction of the plasmon dephasing rate in nanorods compared to small nanospheres due to a suppression of interband damping . These rods show very little radiation damping due to their small volumes. These findings imply large local-field enhancement factors and relatively high light-scattering efficiencies, making metal nanorods extremely interesting for optical applications. Therefore, we are highly interested to examine the possibility of using inorganic fluorescent nanorods, especially lanthanide ortho phosphate LnPO4·H2O [Ln = Eu or Tb], as fluorescent labels in cell biology. On the otherhand, in comparison to organic dyes (including Fluorescein, Texas Red™, Lissamine Rhodamine B, and Tetramethylrhodamine) and fluorescent proteins (Green fluorescent protein, GFP), inorganic fluorescent nanoparticles have several unique optical and electronic properties including size- and composition-tunable emission from visible to infrared wavelengths, a large stokes shift, symmetric emission spectrum, large absorption coefficients across a wide spectral range, simultaneous excitation of multiple fluorescent colors, very high levels of brightness, [4, 13], high resistance to photobleaching, and an exceptional resistance to photo- and chemical degradation [2–5, 13, 16, 17] ].
Bio-conjugated inorganic nanoparticles have raised new possibilities for the ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models, and possibly in human subjects. In this context, lanthanide-based inorganic fluorescents, especially Eu- and Tb-phosphate nanoparticles, have attracted a great deal of attention in cell biology. Optical properties of europium (Eu) and terbium (Tb) salts and their chelates have been used in diverse biomedical applications, namely time-resolved fluorometric assays and immunoassays [18–26]. Furthermore, there are some previous reports regarding the introduction of inorganic luminescent nanospheres such as CdSe, ZnS, PbSe, ZnSe, and ZnS into cells [4, 27, 28]; however, these compounds are toxic to the cells. As the potential toxic effects of nanomaterials (nanospheres or nanorods) is a topic of considerable importance, the in vivo toxicity of Eu and Tb salts will be a key factor in determining whether the fluorescent imaging lanthanide probes could be used in vivo. In our study, lanthanide phosphate [LnPO4·H2O, where Ln = Eu and Tb] nanorods were found to be non-toxic to endothelial cells as analyzed by cell proliferation assays  and the TUNEL assay. Moreover, to the best of our knowledge, there is no known report internalization of naked (nanorods without surface modifications of peptides, organic molecules, or polymers) fluorescent nanorods (EuPO4·H2O and TbPO4·H2O) into cells. In order to functionalize the surface of nanorods, we used aminopropyl trimethoxy silane (APTMS) or mercapto-propyl trimethoxy silane (MPTMS) as reported in the literature . The functionalization of these nanorods using the microwave technique  is currently ongoing in our laboratory.
To the best of our knowledge, this is the first report of inorganic lanthanide phosphate fluorescent nanorods as fluorescent labels in cell biology. In the present study, EuPO4·H2O and TbPO4·H2O nanorods have been prepared by microwave heating and characterized as described previously . The microwave technique is simple, fast, clean, efficient, economical, non-toxic, and eco-friendly . The aim of our study was to investigate whether these inorganic fluorescent nanorods were capable of entering the cells and retaining their fluorescent properties for detection post-internalization. If so, drugs or biomolecules attached to these nanorods can then be detected after internalization and benefit future imaging, therapeutics, and diagnostic purposes. The aim of this paper is not to compare the toxicity of inorganic fluorescent nanorods with other inorganic fluorescent nanoparticles such as CdSe or CdTe but to explore and find new inorganic fluorescent materials that can be used as fluorescent labels in cell biology.
Results and discussion
Excitation and emission spectra of EuPO4·H2O and TbPO4·H2O nanorods were detected at the recommended wavelength by a spectrofluorometer, indicating that properties of the nanorods remained unchanged upon internalization into cells (Fig. 3A–B). However, for confocal microscopy, the same recommended excitation wavelengths were not available on the instrument. Thus, we took confocal images after excitation at 488 nm and collected emission with a 515 nm long pass filter. We found that after excitation at 488 nm and collected the emission spectrum with a 515 nm long pass filter, there was a significant and clear distinction between the fluorescence intensity of untreated cells (Fig. 5A) and nanorod-treated cells (Fig. 5-C). However, after scanning through a number of different excitation wavelengths as reported in the literature , we could not clearly distinguish between the fluorescence intensity of untreated cells and nanorod-treated cells. Because this is our first report using inorganic lanthanide phosphates (EuPO4·H2O and TbPO4·H2O) as a fluorescent biological label, there is no evidence to show that an emission is detectable with a 515 nm long pass filter. However, it was reported in the literature that a 488 nm excitation wavelength  was used in confocal microscopy to detect luminescent properties of europium (III) nanoparticles.
Considering our results from fluorescence spectroscopy, DIC, confocal, and TEM, we've shown that these fluorescent nanorods can be internalized in a cellular system and are readily visualized by microscopy. These nanorods then offer a useful alternative as fluorescent probes for targeting various molecules to specific cells. The exact mechanism for internalization of these nanorods still remains unclear but is under investigation in our laboratory.
To observe viability, HUVEC were treated with 50 μg/ml of europium and terbium phosphate nanorods for 24–48 hours. There was no difference in cell death between untreated control cells (no treatment) and nanorod-treated cells as assessed by trypan blue (data not shown). These results illustrate a biocompatibility between the nanorods and the cells.
Parak et al.  has indicated that the cellular toxicity of stable nanomaterials is primarily due to aggregation rather than the release of Cd elements. However, in our case, since these nanorods are based on an entirely different material than cadmium, their mechanism is likely to be different than Cd-based materials. Therefore, if the toxicity of Cd-based materials is due to an aggregation of ion, that may not be the case for nanorods as supported by our data.
While there is no direct evidence for the effect of nanoparticle size on internalization and toxicity, some reports indicate that nanoparticle size is involved [28, 32, 33]. In our case, we are currently studying in detail the cytotoxicity and mechanism for the cellular internalization of these nanorods. Finally, we should mention in our experiments, the correct control would be a non-fluorescent lanthanide phosphate compound instead of untreated cells. We are currently working on the synthesis of such a reagent. Along with this work, we are also determining: (a) the mechanism of internalization; (b) the cytotoxicity of these materials; (c) the photostability and quantum efficiency of these materials; (d) the surface functionalization of these materials; (e) drug delivery using these nanorods after surface modifications; and (f) the comparison between the fluorescent and non-fluorescent lanthanide phosphate compounds in all experiments.
Nanorods are stable at room temperature indefinitely. We have performed chemical characterizations (XRD, TGA, DSC, TEM, fluorescence properties) on samples that are 4–5 months old and have detected no difference between freshly prepared nanorods and older samples including the absence of any agglomeration.
A novel alternative to conventional organic dyes, we have reported the use of inorganic fluorescent EuPO4·H2O and TbPO4·H2O as a fluorescent label in biomedical research. We have shown internalization of EuPO4·H2O and EuPO4·H2O nanorods by both 786-O cells and HUVEC using fluorescence spectroscopy (FS), DIC, confocal microscopy, and TEM. The nanorods were observed to localize mainly in the cytoplasmic compartments of cells and did not appear to detrimentally affect cell viability nor induce any toxicity after internalization. These unique fluorescent nanorods offer new advancements in the detection and diagnosis for cancer therapy at an early stage and we are currently working on functionalizing these nanorods as well as utilizing them as specific vehicles for drug delivery.
Europium (III) nitrate hydrate [Eu(NO3)3·xH2O, 99.99%], terbium (III) nitrate hexahydrate [Tb(NO3)3·6H2O, 99.999%], ammonium dihydrogenphosphate, [NH4H2PO4 99.999%], were purchased from Aldrich, USA. [3H]-Thymidine was purchased from Amersham Biosciences, Piscataway, NJ. 786-O cells were purchased from American Type Culture Collection (ATCC, TIB-186, Rockville, MD). Dulbeco's Modification of Eagle's Medium (DMEM, 1X) was purchased from Cellgro, Mediatech, Inc, Herndon, VA, USA. Endothelial Cell Basal Medium (EBM), human umbilical vein endothelial cells (HUVEC) were obtained from Cambrex Bio Science alkersvile, Inc, MD, USA.
Microwave-assisted synthesis of lanthanide ortho phosphate hydrates (LnPO4·H2O)
The inorganic fluorescent nanoparticles (LnPO4·H2O) were synthesized using microwave techniques as reported in the literature . In a typical synthesis, 20 ml 0.05(M) of aqueous NH4H2PO4 were added to 20 ml 0.05 (M) of an aqueous solution of Ln(NO3)3 (Ln = Eu and Tb) in a 100 ml round-bottomed flask. The pH of the solution before and after the reaction was in the range of 1.8 – 2.2. The sample was irradiated for 20 min with 50% of the instrument's power. The microwave refluxing apparatus was a modified domestic microwave oven (GOLD STARR 1000W with a 2.45 GHz), described previously . In the post-reaction treatment, the resulting products were collected, centrifuged at 36303 g (20,000 rpm with rav = 8.125 cm), washed several times using ethanol and distilled water, and then dried overnight under vacuum at room temperature. The yield of the as-prepared products is more than 95%.
Cell culture experiments
HUVEC and 786-O cells were cultured at 105 cells/2 ml in six well plates for ~24 h at 37°C and 5% CO2 in EBM and DMEM complete media. For investigating the cellular localization (using confocal microscope), cells were plated on glass cover slips and grown to 90% confluence, and then incubated with LnPO4·H2O nanorods at a concentration of 50–100 μg/ml. After 20 h of incubation, the cover slips were rinsed extensively with phosphate buffered saline (PBS) and cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature and then re-hydrated with PBS. Once all the cells were fixed, the cover slips were mounted onto glass slides with Fluor Save mounting media and examined with DIC and confocal microscopy. For detection of apoptosis using the TUNEL assay (Roche, USA, Cat. No. # 12 156 792 910), cells were mounted onto glass slides with mounting media containing DAPI (4'-6-Diamidino-2-phenylindole).
In another set of experiments, 786-O and HUVEC cells (105 cells/2 ml) were cultured in six well plates and treated with LnPO4·H2O nanorods in corresponding DMEM and EBM complete media without cover slips. After 20 h of incubation with the nanorods, the cells were washed with PBS, trypsinized, and neutralized. The cells were washed by centrifugation and re-suspended in PBS and analyzed with fluorescence spectroscopy, TEM, and flow cytometry (for detection of apoptosis of cells using annexin-FTIC-PI, Bio Vision, USA, catalog # K101-100). Cell viability for another set of cells was determined through staining with trypan blue and cells were counted using a hemocytometer.
Cell proliferation assay
Cell proliferation to measure in vitro toxicity was performed with the [3H]-thymidine incorporation assay according to the reported literature . Briefly, endothelial cells (HUVEC; 2 × 104) were seeded in 24-well plates, cultured for 2 days in EBM, serum-starved (0.1% serum) for 24 hours, and then treated with different concentrations (1–100 μg/mL) of LnPO4·H2O (Ln = Eu, Tb). After 20 hours, 1 μCi [3H] thymidine was added to each well. Four hours later, cells were washed with cold PBS, fixed with 100% cold methanol, and collected for the measurement of trichloroacetic acid-precipitable radioactivity . Experiments were repeated in triplicate and all results were reproducible.
Cells were seeded into 6-well plates at a density of 2 × 105 /2 ml of medium per well and grown overnight. After appropriate treatment with these nanorods (50 μg/mL), cells were extensively washed with PBS and tested with the Annexin V-FITC Apoptosis Detection Kit (Biovision, Cat. No. #K101-100) per the manufacturer's instructions. In addition, apoptosis was also determined by the TUNEL assay using the In Situ Cell Death Detection Kit, TMR red (Roche, Cat. No. #12 156 792 910). The red apoptotic cells were visualized on a microscope, counted (6 fields per sample), and photographed using a digital fluoresence camera.
Transmission electron microscopy study
Particle morphology (microstructures of the samples) was studied with TEM on a FEI Technai 12 operating at 80 KV. To visualize the internalization of particles inside the cells, we have folllowed the published literature procedures [35, 36].
The excitation and emission (fluorescence) spectra were recorded on a Fluorolog-3 Spectrofluorometer (HORIBA JOBINYVON, Longjumeau, France) equipped with a xenon lamp as the monochromator excitation source.
Differential interference contrast microscopy (DIC)
After fixation of cells on cover slips, the cells were mounted onto glass slides with Fluor Save mounting media and examined for DIC. Pictures were captured with AXIOCAM high-resolution digital camera using an AXIOVERT 135 TV microscope (ZEISS, Germany).
Confocal fluorescence microscopy
Two dimensional confocal fluorescence microscopy images were collected through use of LSM 510 confocal laser scan microscope (Carl Zeiss, Inc., Oberkochcn, Germany) with C-Apochromat 63 X/NA 1.2 water-immersion lense, in conjunction with an Argon ion laser (488 nm excitation). The fluorescence emissions were collected through a 515 nm long pass filter.
After mounting the cells onto glass slides with DAPI, images were collected through a LSM 510 confocal laser scan microscope (Carl Zeiss, Inc., Oberkochcn, Germany) with a C-Apochromat 63 X/1.2 na water-immersion lens. The fluorescence emissions were collected through a 385–470 nm band pass filter in conjunction with an argon ion laser excitation of 364 nm for DAPI. The fluorescence emissions were collected through a 560–615 nm band pass filter in conjunction with a HeNe1 ion laser excitation of 543 nm for TMR red.
We are thankful to Drs. William Wessels, Franklyn Prendergast, Sutapa Sinha, Kaustubh Datta, Michael Muders, and Enfeng Wang for their scientific help and discussion. We are also thankful to J. Tarara and J. Charlesworth for their help with the confocal and transmission electron microscopy, respectively. This work was partly supported by NIH grants CA78383 and HL70567 and also a grant from the American Cancer Society to D. Mukhopadhyay.
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