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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Departments of Neuroradiololgy (S.H.) and Pathology (U.V.), University of Tübingen, Tübingen, Germany
Received June 23, 2006; accepted August 18, 2006.
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Abstract |
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) and treatment (e.g., neutron capture therapy) (Martin et al., 1989; de Stasio et al., 2001)] would require the accumulation of a Gd-DTPA conjugate in the cell nucleus. Therefore, it is a matter of the utmost importance to develop small Gd conjugates that are able to cross the cell membrane without the need for large transmembrane transport peptides and can also enter the cell nucleus.
Nuclear localization sequences (NLSs) are short peptides that enable cytoplasmic proteins to enter the cell nucleus (Jans, 1995). Fluorescein isothiocyanate (FITC)-labeled NLSs can cross not only the nuclear membrane but also the cell membrane when added to the cell culture medium (Ragin et al., 2002).
We have developed a Gd conjugate composed of the gadolinium complex linked with two different NLSs (Scheme 1): the NLS of the simian virus (SV) 40 T antigen (Kalderon et al., 1984) and the NLS of the ALL-1 protein (Yano et al., 1997), containing motifs of several other NLSs [e.g., arginine-lysine-arginine of p21 (Rodriguez-Vilarrupla et al., 2002), arginine-lysine-arginine-lysine-arginine of p45TC (Lorenzen et al., 1995), or lysine-arginine-lysine-arginine of PLAG1 (Braem et al., 2002)].
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-sheet conformation, are predominantly responsible for the interaction of penetratin with the negatively charged phospholipids of the cell membrane, leading to cellular uptake (Binder and Lindblom, 2004; Christiaens et al., 2004).
Of course, an alternative would have been to use large polyarginine or polylysine chains, but such peptide sequences induce microvascular leakage of macromolecules in animals, polyarginine representing a surrogate for eosinophil basic proteins (Rosengren and Arfors, 1991; Minnicozzi et al., 1995). Therefore, we chose to use a short peptide sequence consisting of alternating arginines and lysines.
FITC was bound to the Gd conjugate to enable confirmation of nuclear localization by fluorescence microscopy. The conjugate was injected i.p. and i.v. in nude mice bearing human LN-229 glioma xenografts.
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Materials and Methods |
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; Carpino and Han, 1972) in a fully automated synthesizer (ABI 431) (Applied Biosystems, Weiterstadt, Germany) on 0.05 mmol of Fmoc-Lys(Boc)-tritylchloride polystyrol resin (Trityl-Resin) (PepChem Tübingen, Germany) and on a 0.052 mmol Fmoc-Glu(t-butoxy)-tritylchloride polystyrol resin (Trityl-Resin). As a coupling agent, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (Novabiochem, Bad Soden, Germany) was used. The side chains of trifunctional amino acids were protected as follows: Arg(2,2,4,6,7 pentamethyldihydro-benzofuran-5-sulfonyl), Lys(Boc), Lys(4-methoxytrityl), Lys(ivDde), and Glu (t-butoxy) (Novabiochem). We used Boc-Pro-OH (Novabiochem) for coupling of the N-terminal amino acid. The protected peptidyl resin was treated with 20% hexafluoroisopropanol (Merck, Darmstadt, Germany) in dichloromethane (SDS, Peypin, France) for 5 to 10 min and yielded a fully protected peptide. The 4-methoxytrityl-protecting group was cleaved by treating it for 3 x 5 min in 1% trifluoroacetic acid (Applied Biosystems) in dichloromethane and followed by coupling with FITC (Novabiochem). The ivDde-protecting group was cleaved with 4% hydrazine (Fluka, Buchs, Switzerland) in dimethylformamide (SDS). The DTPA was added as dianhydride (Merck), and the coupling reaction lasted 6 h.
The protection of the DTPA peptide was removed by treatment with 90% trifluoroacetic acid, 5% ethanedithiol (Fluka), 2.5% thioanisol (Sigma-Aldrich, Taufkirchen, Germany), and 2.5% (v/v) phenol (Fisher Scientific, Schwerte, Germany) for 2.5 h at room temperature. The products were precipitated in ether. The crude material was purified by preparatory high-performance liquid chromatography (Shimadzu LC-8A; Shimadzu, Kyoto, Japan) on a YMC-Pack ODS-AQ 120 Å, S-5-µm reverse phase column (20 x 150 mm) using an eluent of 0.1% trifluoroacetic acid in water (A) and 60% acetonitrile (Fisher Scientific) in water (B). The peptide was eluted with a successive linear gradient of 25% B to 60% B in 40 min at a flow rate of 20 ml/min. The fractions corresponding to the purified protein were lyophilized.
The peptide was dissolved in a small amount of distilled water and added drop-wise under stirring to a gadolinium solution [gadolinium (III) chloride hexahydrate; Sigma-Aldrich] for a period of 1 h. The pH was maintained at 5.8 by adding 0.1 M NaOH. The solution was dialyzed until no free gadolinium was detected, using Xylenol-Orange (Fluka) as indicator. The purified material was characterized by using analytical high-performance liquid chromatography (Shimadzu LC-10; Shimadzu) and matrix-assisted laser desorption mass spectrometry (Finnegan MAT Vision 2000; Thermo Electron Corporation, San Jose, CA). Substance purity was 99%.
Tumor Implantation
The animal experiments were approved by the Committee for Animal Experiments of the Regional Council (Regierungspräsidium), Tübingen, and have been carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Athymic female nude mice (CD1 Nu/Nu) (weight, 25 g; age, 7 weeks) were purchased from Charles River (Sulzfeld, Germany). Human LN-229 glioma cells were maintained and implanted intracerebrally as described elsewhere (Friese et al., 2003). Investigations with the Gd conjugate were performed 4 weeks after tumor implantation.
Influx Studies
The mice were subjected to i.p. injection of 0.5 ml of isotonic saline (two animals), 0.5 mg of FITC in 0.5 ml of isotonic saline (two animals), or 4 mg of conjugate in 0.5 ml of isotonic saline (four animals) and killed after 60 min. The organs and brain tumors were excised and processed, as described below.
Efflux Studies
To determine whether the conjugate also passes out of the cell nucleus, it was injected i.p. into six animals, which were killed after 40 min (two animals), 2.5 h (two animals), or 3 days (two animals). The animals were not anesthetized at the time of injection or during the period of observation so that their behavior could be evaluated.
MRI Measurements
The animals were sedated by i.p. injection of ketamine (100 mg/kg) plus xylazine (10 mg/kg). They subsequently received an injection of 4 mg of conjugate in 0.5 ml of isotonic saline (i.p. administration; three animals) or 3 mg of conjugate in 0.25 ml of isotonic saline (slow i.v. administration via the tail vein; three animals). MRI was then performed using a clinical 3-Tesla Siemens whole-body MRI (Magnetom TRIO; Siemens, Erlangen, Germany) with the mice in prone position in a standard circular polarized wrist coil.
Imaging Protocol for Native and Postcontrast Scans
Three-Dimensional Turbo Spin Echo Sequence. Slice thickness was 0.3125 mm, field of view read was 63 mm, field of view phase was 100.0%, base resolution was 256, phase resolution was 100%, slice resolution was 100%, voxel seize was 0.2 x 0.2 x 0.3 mm, slab group was 1, slabs were 1, slices per slab were 16, TR was 300 ms, TE was 15 ms, flip angle was 70, distance factor was 50, and scan time was 12:02 min.
T1 Weighted Transverse Images. Slice thickness was 2 mm, field of view read was 31 mm, field of view phase was 82.3%, voxel seize was 0.2 x 0.2 x 2 mm, TR was 600 ms, TE was 18 ms, flip angle was 180, number of slices was 12, distance factor was 0, and scan time was 11:35 min.
Thirty minutes after i.v. injection and 1 h after i.p. injection, the animals were killed, and the organs and brain tumors were excised and processed, as described below. MRI and confocal laser scanning microscopy (CLSM) scans of the tumors and organs were also obtained 30 min after i.v. injection of 0.25 ml of isotonic saline alone (two animals) and 1 h after i.p. injection of 0.5 ml of isotonic saline alone (two animals). These control animals were subsequently killed, and the organs were excised.
Techniques to Evaluate Nuclear Uptake
In Vivo Fluorescence Microscopy. Before injection of the conjugate (three animals) and isotonic saline alone (one animal), respectively, the skin of the nude mice was examined for green fluorescence using a fluorescence microscope (Axioskop; Carl Zeiss, Jena, Germany) with appropriate filters and beam splitters and an illuminator (N HBO103; Carl Zeiss). Pictures were taken with a 3-CCD color video camera (MC3254P; Sony, Tokyo, Japan) and the Axiovision software (Carl Zeiss). This examination was repeated 45 min after the injection to demonstrate peritoneal uptake and subsequent systemic distribution of the conjugate. The peritoneal cavity was then opened, and the mesentery was examined for nuclear fluorescence using the fluorescence microscope.
Touch Prints. The organs were sliced immediately after excision for the preparation of touch print specimens, which were air-dried and examined with the fluorescence microscope.
Fresh-Frozen Sections. Organ specimens were also snap-frozen in Tissue Tek OCT in liquid nitrogen. Frozen sections (4 µm) were prepared and evaluated using the fluorescence microscope and a confocal laser scanning microscope (LSM 410, Axiovert 135 M; Carl Zeiss). Cell nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes, Eugene, OR). For the fluorescence excitation of FITC and TO-PRO, we used the 488- and the 633-nm lines, respectively, of an argon ion and helium-neon laser, respectively, and appropriate beam splitters and barrier filters. Superimposed images of FITC- and TO-PRO-stained samples were created by overlaying coincident views. H&E-stained sections of these specimens were also prepared.
Semithin Sections. Organ specimens were also fixed in paraformaldehyde, dehydrated in ethanol with progressive lowering of the temperature, embedded in Lowicryl K4M (Polysciences, Eppelheim, Germany), and UV-polymerized at –30°C according to the manufacturer's instructions. Semithin sections (approximately 0.4 µm) were examined with the fluorescence microscope.
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In addition, immunoelectronmicroscopy was performed using an 18-nm gold-labeled anti-FITC antibody (Dianova, Hamburg, Germany). Specimens were analyzed and documented using an EM 10A electron microscope (Zeiss, Oberkochen, Germany).
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Results |
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The conjugate was also detectable by electron microscopy and MRI after i.p. and i.v. injection [increase in magnetic resonance signal intensity in the brain tumors and all the organs except for healthy brain parenchyma with an intact blood-brain barrier (Fig. 3a and Supplemental Data Fig. 1)] and was rapidly excreted via the bile and urine.
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Discussion |
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). However, it would not be possible to apply these concentrations and incubation times in vivo, so that this method does not seem to be a practicable possibility.
Gd-tetraazacyclododecanetetraacetic acid complexes conjugated with HIV-1 Tat-derived membrane translocation peptides have been shown to be taken up by the nuclei of mammalian cells (Bhorade et al., 2000; Prantner et al., 2003). However, the HIV-1 Tat protein induces apoptosis of hippocampal neurons in rats by a mechanism involving caspase activation (Kruman et al., 1998). Mice that had been given HIV-1 Tat Gd-tetraazacyclododecanetetraacetic acid conjugates i.p. died 90 min later (Prantner et al., 2003).
In vitro nuclear uptake of the Gd-DTPA complex has also been accomplished by coupling to the SV-40 T antigen NLS via a noncleavable spacer. The NLS was, in turn, cleavably linked to a penetratin-like transmembrane transport peptide via a disulfide bond. However, it was found that the conjugate cannot leave the cell nucleus (Heckl et al., 2002). Motexafin gadolinium, a synthetically expanded porphyrin containing gadolinium, was found to be taken up by at least 90% of glioblastoma cell nuclei after a long incubation time of 72 h (de Stasio et al., 2006). However, only 15% of murine EMT 6 mammary sarcoma cell nuclei were stained after incubation for 45 h, and no nuclear uptake of motexafin gadolinium was achieved in murine Rif-1 fibrosarcoma cells (Woodburn, 2001).
We report findings concerning the nuclear influx and efflux of a novel Gd conjugate in a nude mouse model. The conjugate, produced by the binding of two NLSs to the gadolinium complex, could be transported not only across the nuclear membrane, as expected, but also across the cell membrane. Interestingly, large transmembrane transport units like penetratin (Heckl et al., 2002) could be omitted, resulting in a comparatively small conjugate with a proportionally low peptide content for each gadolinium complex, which is the most important constituent of the conjugate, producing as it does the signal on MRI and representing the target for possible neutron capture therapy. It is unclear whether our conjugate of 3 kDa was taken up by the nucleus via an active transport mechanism (e.g., importin 
or ) or passive diffusion. It is thought that small molecules (<10 kDa) can pass freely through the nuclear pores (Wei et al., 2003). However, a Gd conjugate of comparable size (Gd complex bound to 16 arginine, approximately 3 kDa) (Allen and Meade, 2003) was found to remain in the cytoplasm and could not enter the nucleus.
To exclude the possibility of fixation-based artifactual relocation of the Gd conjugate to the cell nucleus, as demonstrated for small peptides (Richard et al., 2003), we used various different techniques that avoid the need for fixation: in vivo fluorescence microscopy and methods employing touch prints and fresh-frozen sections. These techniques produced the same results as investigations performed on semithin sections of fixed tissue.
The Gd conjugate described in this study enters a range of different cell types nonspecifically. However, to be of use in tumor therapy, targeting of specific cells by the conjugate would be desirable. It may be possible, for example, to construct Gd conjugates that are taken up by cell nuclei only in the presence of certain extracellular enzymes (e.g., matrix metalloproteinase-2) expressed predominantly by tumors, so that tumor-directed therapy can be achieved (Jiang et al., 2004). In addition, studies need to be performed to evaluate whether similar results could be achieved with smaller Gd conjugates containing only one NLS.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Gd-DTPA, gadolinium-diethylenetriamine pentaacetic acid; MRI, magnetic resonance imaging; NLS, nuclear localization sequence; FITC, fluorescein isothiocyanate; SV, simian virus; Fmoc, 9-fluorenylmethoxycarbonyl-t-butyl; Boc, t-butyloxy-carbonyl; ivDde, 4,4-dimethyl-2,6-dicyclohexyl-1-xylidine-3-methylbutyl; CLSM, confocal laser scanning microscopy; TR, time to repetition; TE, time to echo; ALL-1, acute lymphatic leukemia.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Stefan Heckl, Department of Neuroradiology, University of Tübingen, Medical School, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. E-mail: stefan.heckl{at}med.uni-tuebingen.de
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