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Lanthanide phosphate nanorods as inorganic fluorescent labels in cell biology research.

One of the fundamental goals in biology is to understand the complex spatiotemporal interplay of biomolecules from the cellular to the integrative level. To study these interactions, researchers commonly use fluorescent labeling for both in vivo cellular imaging and in vitro assay detection (1, 2). In this context, one of the fastest developing and most exciting interfaces of nanotechnology is the use of inorganic quantum dots or fluorescent nanoparticles in cell biology. The unique optical properties of inorganic particles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In addition, the ability to make such nanoparticles and then target these particles to specific biomolecules has led to promising applications in cellular labeling, deep-tissue imaging, and assay labeling, and also as efficient fluorescence resonance energy transfer donors.

Conventionally used fluorescent labels, such as organic dyes in cell biology, are prone to problems such as broad spectral features, short lifetime, photobleaching, and potential toxicity to the cells. Inorganic fluorescents, especially europium (Eu) and terbium (Tb) in the lanthanide group, have several unique optical and electronic properties, such as size- and composition-tunable emission from visible to infrared wavelengths, large absorption coefficients across a wide spectral range, symmetric emission spectrum, a large Stokes shift, simultaneous excitation of multiple fluorescent colors, very high levels of brightness, and photostability (3-5). Furthermore, analysis by cell proliferation and apoptosis assay (dUTP nick-end labeling) showed that lanthanide phosphate (Ln[P0.sub.4] * [H.sub.2]0, where Ln = Eu) and Tb nanorods synthesized by microwave (MW) were nontoxic to endothelial cells (5).

In this study, we synthesized Ln[P0.sub.4] * [H.sub.2]O nanorods by an exclusive MW technique and used a transmission electron microscope (TEM) to investigate the internalization of Ln[P0.sub.4] * [H.sub.2]O nanorods into 786-0 cells and human umbilical vein endothelial cells (HUVECs). Confocal microscopy results showing improved fluorescence imaging (red fluorescence for Eu, green fluorescence for Tb, and no autofluorescence for controls) suggest that this technique might be used for live-cell imaging, a requisite analytical tool in most cell biology experiments and a routine procedure in neurobiology, developmental biology, pharmacology, and several other related biomedical research fields.

Eu(III) nitrate hydrate, Tb(III) nitrate hexahydrate, and ammonium dihydrogenphosphate were purchased from Aldrich and used without further purification. 786-0 cells were purchased from ATCC, and HUVECs were obtained from Cambrex.

The inorganic fluorescent nanorods (Ln[P0.sub.4] * [H.sub.2]O) were synthesized and characterized using MW techniques as we previously reported (6). Briefly, 20 mL of a 0.05 mol/L aqueous NH4HZPO4 solution was added to 20 mL of a 0.05 mol/L aqueous solution of Ln([N0.sub.3]).sub.3] in a 100-mL round-bottomed flask (see the Data Supplement that accompanies the online version of this article at http: / /www. The yield of the as-prepared products was more than 95%.

Ln([N0.sub.3).sub.3] + [NH.sub.4][H.sub.2][PO.sub.4] + [H.sub.2]O + MW-heating[right arrow] Ln[P0.sub.4]-[H.sub.2]0

The MW-prepared Ln[P0.sub.4] * [H.sub.2]O products were pure and crystallized with high yield. The technique did not require high temperatures, high pressure, a catalyst, reduced pressure conditions, or preprocessing.

This method is easy to perform, fast, clean, efficient, and nontoxic. The MW technique is considered to be ecologically friendly for the following reasons: (a) the speed of chemical reactions is enhanced with minimal energy output and thus the heating energy is very focused; in contrast, the use of a heating plate wastes more energy; (b) 2.45-GHz radiation is not harmful; and (c) the solvents used, such as water and ethanol, efficiently absorb MW radiation and are also considered environmentally friendly. The as-synthesized Eu[P0.sub.4] * [H.sub.2]O products consisted of nanorods (6 to 8 run in diameter and 100 to 300 run long), and Tb Eu[P0.sub.4] * [H.sub.2]O products were a mixture of 2 rod types in micrometer sizes (small rods 0.5 to 1.5 [micro]m long and 6 to 8 run wide, and bigger rods 1.1 to 2.2 [micro]m long and 80 to 130 run wide) (5).


We prepared the Ln[P0.sub.4] * [H.sub.2]O nanorod suspension in sterile Tris-EDTA buffer and used only freshly prepared suspension for all cell culture experiments. HUVEC and 786-0 cells were cultured at 105 cells/2 mL in 6-well plates for 20 h at 37[degrees]C and 5% [C0.sub.2] in endothelial cell basal medium and DMEM, respectively. For detection of cellular localization of Ln[P0.sub.4] * [H.sub.2]O nanorods, after 20 h of incubation with these nanorods, the cells were washed with PBS [[KH.sub.2][PO.sub.4] (0.144 g/L; 1.06 mM), NaCl (9 g/L; 153.8 mNl), [Na.sub.2][HP0.sub.4] (anhydrous) (0.795 g/L; 5.6 mM), pH 7.4], trypsinized, neutralized, and washed by centrifugation. The cells were then resuspended in fixative liquid and analyzed with a TEM according to the accepted protocol (7,8). For investigation of fluorescence images (by use of confocal microscopy), cells were plated on glass coverslips and grown to 90% confluence, after which they were incubated with Ln[P0.sub.4] * [H.sub.2]O nanorods at a concentration of 50100 mg/L. After 20 h of incubation, the coverslips were rinsed extensively with PBS; cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature and then rehydrated with PBS. When all the cells were fixed, the coverslips were mounted onto glass slides with Fluor Save mounting media and examined with confocal microscopy. The minimum concentration of the nanorods used for internalization in the RCC/HUVEC and visualization by transmission electron microscopy/confocal microscopy was 10 mg/L (data not shown).

The internalization of Ln[P0.sub.4] * [H.sub.2]O nanorods inside the cells was visualized with a TEM according to previously published procedures (7,8). The TEM image of 786-0 cells (after cross-section) treated with [P0.sub.4] * [H.sub.2]O nanorods, at concentrations of 50 mg/L with different magnifications, clearly visualized the nanorods inside the cytoplasm of the cells (see Fig. SI-1 in the online Data Supplement). Interestingly, both Eu[P0.sub.4] * [H.sub.2]O and Tb[P0.sub.4] * [H.sub.2]O nanorods remain unchanged after internalization into the cytoplasm of the cell. The exact nature of the cytoplasmic vesicles is currently under investigation as part of a future project, but it appears that nanorods internalize in double-membrane layered early endosomelike compartments away from the nucleus or late endosomelike structures close to the nucleus.

Two-dimensional, confocal fluorescence microscopy images were collected through the use of an LSM 510 confocal laser scan microscope (Carl Zeiss) with a C-Apochromat 63 X/NA 1.2 water-immersion lens in conjunction with an argon ion laser (364 and 488 run excitations were used for Eu and Tb, respectively). The red and green fluorescence emissions were collected through a 601- and 505-nm long-pass filter for Eu[P0.sub.4] * [H.sub.2]O and TbEu[P0.sub.4] * [H.sub.2]O nanorods, respectively, and are shown in Fig. 1, A and C. A red fluorescence (Fig. 1A) of EU[P0.sub.4] * [H.sub.2]O nanorods and its corresponding phase image (Fig. 113) are observed in Fig. 1, A and B. Similarly, a green fluorescence (Fig. 1C) of Tb[P0.sub.4] * [H.sub.2]O nanorods and its corresponding phase image (Fig. 1D) are shown in Fig. 1, C and D. Furthermore, the fluorescence (Fig. 1, E, G, and I) and corresponding phase image (Fig. 1, F, H, and J) of the control untreated 786-0 cells and 786-0 cells treated with Ln[P0.sub.4] * [H.sub.2]O nanorods are presented in Fig. 1, E -J. There are no Ln[P0.sub.4] * [H.sub.2]O nanoparticles observed in the phase image of control untreated 786-0 cells in Fig. 1F and no fluorescence (even autofluorescence) in Fig. 1E, which clearly indicates the absence of Ln[P0.sub.4] * [H.sub.2]O nanorods. On the other hand, a very nice and clear red fluorescence in Fig. 1G was observed due to the presence of Eu[P0.sub.4] * [H.sub.2]O nanorods inside 786-0 cells. Similarly, very nice and bright green fluorescence in Fig. 11 indicates the presence of Tb[P0.sub.4] * [H.sub.2]O nanorods inside 786-0 cells. Overall, there is a significant difference in red (Fig. 1G) and green (Fig. 11) fluorescence between the untreated control cells (Fig. 1E) and the nanorod-treated cells (Fig. 1, G and n. Similar results were obtained for HUVECs after treatment with Ln[P0.sub.4] * [H.sub.2]O nanorods (see Fig. SI-2 in the online Data Supplement). These results demonstrate the internalization of Ln[P0.sub.4] * [H.sub.2]O nanorods inside 786-0 and HUVECs. One of the most important results was that under the new settings of the confocal microscope, we were able to clearly distinguish the great difference in red and green fluorescence intensities between untreated control cells and nano-rod-treated cells, whereas in our earlier study, autofluorescence was present in control untreated cells (5).

Taken together, these results indicate that these fluorescent nanorods can internalize in cells, which in turn can be visualized by microscopy. Therefore, these nanorods offer useful and alternative inorganic fluorescent probes for targeting various molecules in living cells. Furthermore, we also report the use of inorganic fluorescent Eu[P0.sub.4] * [H.sub.2]O, Tb[P0.sub.4] * [H.sub.2]O nanorods as a fluorescent label (a novel alternative to conventional organic dyes) in biomedical research. We have shown, by use of confocal microscopy and transmission electron microscopy, internalization of Eu[P0.sub.4] * [H.sub.2]O, Tb[P0.sub.4] * [H.sub.2]O nanorods into 786-0 and HUVECs. These nanorods were observed to localize mainly in the cytoplasm of these cells and did not appear to detrimentally affect cell viability or induce toxicity after internalization.

Finally, our inorganic fluorescent label method is a simple tool for examining the cellular compartments of living cells. Fluorescent nanorods may enable improved detection of malignant tumor cells.

Grant/funding support: This work was partly supported by National Institutes of Health Grants CA78383 and HL70567, a grant from the American Cancer Society (to D.M.), a developmental grant from the Haem-Malignancy Program at the Mayo Clinic (RAF-20P), and a CLL-Global Foundation grant (to P.M.).

Financial disclosures: None declared.

Acknowledgments: We are thankful to Drs. William Wessels, Franklyn Prendergast, Kaustubh Datta, and Michael Muders for scientific help and discussion. We are also thankful to Jim Tarara and John Charlesworth for help with the confocal and transmission electron microscopy, respectively.

DOI : 10.1373/clinchem.2007.091207


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Chitta Ranjan Patra, Resham Bhattacharya, Sujata Patra, Sujit Basu, Priyabrata Mukherjee, and Debabrata Mukhopadhyay *

(Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic, Rochester, MN;

* address correspondence to this author at: Departments of Biochemistry and Molecular Biology, and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905; fax 507-284-1767, e-mail
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Title Annotation:Abstracts of Oak Ridge Posters
Author:Patra, Chitta Ranjan; Bhattacharya, Resham; Patra, Sujata; Basu, Sujit; Mukherjee, Priyabrata; Mukho
Publication:Clinical Chemistry
Date:Nov 1, 2007
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