Macrocyclic Chelators with Paramagnetic Cations Are Internalized into

MAY/JUNE 2000 Volume 11, Number 3 © Copyright 2000 by the American Chemical Society

COMMUNICATIONS Macrocyclic Chelators with Paramagnetic Cations Are Internalized into Mammalian Cells via a HIV-Tat Derived Membrane Translocation Peptide Rajeev Bhorade, Ralph Weissleder, Tsunenori Nakakoshi, Anna Moore, and Ching-Hsuan Tung* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, 149 13th St., #5406, Charlestown, Massachusetts 02129. Received December 7, 1999

A major obstacle to using paramagnetic MR contrast agents for in vivo cell tracking or molecular sensing is their generally low cellular uptake. In this study, we show that a paramagnetically labeled DOTA chelator derivatized with a 13-mer HIV-tat peptide is efficiently internalized into mammalian cells. Intracellular concentrations were attained that were readily detectable by MR imaging using both gadolinium and dysprosium chelates. Using this paradigm, it should be feasible to internalize a variety of chemically different agents into mammalian cells.

INTRODUCTION

The cellular plasma membrane represents a natural barrier to many exogeneously administered molecules. To overcome this delivery barrier for therapeutic and diagnostic drugs, a variety of strategies have been proposed to ferry agents through cellular membranes. These strategies include receptor mediated uptake (1, 2), fluid phase endocytosis (pinocytosis) (3), direct fusion with the membrane lipid bilayer (4), and use of peptide/ protein mediated translocation signals (5, 6). Several membrane translocation signal (MTS) peptides have recently been reported, including the HIV-tat (7, 8), the third helix of the homeodomain of Antennapedia (9), a peptide derived from anti-DNA monoclonal antibody (10), VP22 herpes virus protein (11, 12), and other synthetic peptides (13, 14). Although the mechanism of entry into * To whom correspondence should be addressed. Phone: (617) 726-5788. Fax: (617) 726-5708. E-mail: tung@ helix.mgh.harvard.edu.

the cells often remains unknown, MTS have been used to internalize proteins (7, 15-20), peptides (8, 21, 22), antisense oligonucleotides (23, 24), peptide nucleic acid (25), and plasmid DNA (26-28). Recently, we demonstrated the MTS peptide from the HIV-tat peptide can be used to induce nonphagocytic cells to internalize a diagnostic label often used in MR imaging, superparamagnetic iron oxide particles (29). When attached to the HIV tat peptide, nanometer-sized superparamagnetic iron oxide crystals were internalized by HeLa cells, murine lymphocytes, and human NK cells to levels that made the cells detectable by MR imaging. However, superparamagnetic labels can potentially produce susceptibility artifacts and in their intracellular form have little effect on T1 weighted MR images. In addition, a variety of “smart” paramagnetic MR contrast agents are recently under development that alter their T1 relaxivity when conformational changes occur, thus allowing molecular sensing (30, 31). However, to date one of the main problems of using these reporters is their

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poor cell permeability. We hypothesized that macrocyclic chelators could be conjugated to an HIV-tat peptide and thus synthesized a peptide and 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) conjugate. By attaching paramagnetic chelates to the HIV-tat peptide, we show for the first time highly efficient cell uptake and demonstrate these effects by MR imaging of cells. These results suggest a variety of diagnostic agents could be synthesized that will readily cross cell membranes. RESULTS AND DISCUSSION

The translocating properties of the tat peptide (GRKKRRQRRR) representing residues 48-58 of the HIV tat protein has been demonstrated by several groups (15, 17, 32). The peptide efficiently localizes to cytoplasmic and nuclear compartments in a time-dependent fashion and seems to have also a high propensity for nucleosomes in several cell types. In one prior study, we have shown that 40 nm particles could be internalized into the nuclear compartment when conjugated to the tat peptide (29). In the current study, we conjugated a macrocyclic chelator, DOTA, to the C-terminus of the tat peptide and then complexed with different metal ions. The peptide, GRKKRRQRRRGYK(DOTA)-NH2 (tat-DOTA), was synthesized using solid-phase synthesis on an automatic peptide synthesizer, as illustrated in Figure 1. An orthogonal approach was applied to result in monodeprotection of the C-terminal lysine side chain. A protected DOTA analogue, 1,4,7,10-tetraazacyclododecane-1,4,7tris(acetic acid tert-butyl ester)-10-acetic acid (DOTA3tBu) (Macrocyclics, Richardson, TX), was activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) and coupled to the designated position. The subsequent trifluoroacetic acid treatment not only cleaved the peptide off the resin but also removed all protecting groups from the peptide as well as DOTA-3tBu. The peptide was purified by reversed-phase high-pressure liquid chromatography (HPLC) and characterized by mass spectrum, MALDI-MS; (M + H)+: 2131.4 (calcd), 2131.3 (found). The chelation of gadolinium (Gd) and dysprosium (Dy) was performed by incubating tat-DOTA with GdCl3 and DyCl3, respectively, in glycine/HCl buffer 50 mM, pH 3.5 at 80 °C for 3 h. The conjugates were further purified by reversed-phase HPLC. MALDI-MS (M + H)+: tat-DOTAGd, 2285.7 (calcd), 2285.6 (found); tat-DOTA-Dy 2290.9 (calcd), 2291.3 (found). To monitor the cellular distribution of the peptide, a modified peptide was prepared using the same synthetic approach, except that DOTA-3tBu coupling was replaced with fluorescein isothiocyanate (FITC). The sequence of the latter peptide was GRKKRRQRRRGYK(FITC)-NH2, and its translocation properties were studied in human epithelial HeLa cells. At 37 °C, a pronounced nuclear accumulation was observed within 30 min (Figure 1). A similar distribution has also been described by several other investigators (7, 8, 29). The precise mechanism of uptake has so far not been elucidated but adsorptive endocytosis has been proposed (33). Cellular uptake of the tat-DOTA conjugate was quantitated using the 111In-labeled tat-DOTA conjugate. TatDOTA labeling was performed by adding 50 µCi of 111InCl to 5 µmol of tat-DOTA in 1 mL of 50 mM glycine/ 3 HCl buffer, pH 3.5 at 80 °C for 3 h. The chelation efficiency, determined by analytic reversed-phase HPLC with an ratioactivity detector, was greater than 99%. Fresh lymphocytes (1 × 106 cells) were then obtained from mouse spleen (34) and were incubated with various

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amounts of tat-DOTA-111In (25, 50, 100, 150, or 200 nmol) in 1 mL of RPMI 1620 medium at 37 °C for 60 min. After incubation, cells were washed three times with Hanksbuffered saline solution and then resuspended in RPMI 1620 medium. In parallel, DOTA-111In without tat peptide was used as a control. As shown in Figure 2A, uptake increased with increasing amounts of tat-DOTA conjugate without reaching saturation at 200 nmol of peptide per million cells. In contrast, uptake of DOTA-111In was negligible at all concentrations. Splenocytes, previously labeled with 100 nmol of InCl3, were kept in RPMI 1620 medium for 2 days while the medium was replaced daily. The intracellular tat-DOTA retention was determined by radioactivity counting, and adjusted for cell numbers. At 0, 24, and 48 h, 2.6, 2.2, and 2.5 fmol/cell were found, respectively. This result suggests that the tat peptide is required for translocation, and once the compound is internalized, it will stay inside for several days. In another experiment, cells were incubated with 40 nmol of tat-DOTA-111In (20 µCi) for 15, 30, 45, or 60 min. The time curve of cellular uptake is shown in Figure 2B. Uptake was relatively rapid with 75% of maximum cell uptake occurring within 15 min of incubation. Additional experiments were also performed at different temperatures (4 and 37 °C), which showed similar uptake kinetics, suggesting that the mechanism of uptake is not receptor mediated as reported by other groups (7, 8). A cytotoxicity assay was used to determine whether tat-DOTA analogues would be toxic to lymphocytes. Cells (1 × 106) were incubated with 10 or 200 nmol of tat-DOTA-Gd for a period of 5 days, and the cell viability was determined daily by cell counts. About 80% of cells remained alive at all concentrations tested, and similar viability figures were found in the control groups. To determine whether cell internalized paramagnetic tat-DOTA conjugates would be detectable by MR imaging, we performed the following experiment. Freshly isolated murine lymphocytes (106 cells) were labeled with either 100 nmol of tat-DOTA-Gd or tat-DOTA-Dy for a period of 1 h in RPM1 medium. After incubation, cells were washed three times with Hanks buffered saline solution as described previously. The labeled cells were embedded in agar cylinders to minimize air interface artifacts. MR imaging was performed with a 1.5 T superconducting magnet (Signa 5.0; GE Medical Systems, Milwaukee, WI) using a 5-in. surface coil. The time between labeling of cells and imaging was approximately 12 h. The imaging protocol consisted of coronal T1 weighted spin-echo (SE 300/11 where the first number represents the repetition time, TR, and the second number represents the echo time, TE), T2 weighted SE (SE 2000/102) or T2* gradient echo sequence (SteadyState Free Precession pulse sequence with TR/TE 50/20 and a flip angle of 20°) representative of commonly used pulse sequences in clinical imaging. Slice thickness was 1-3 mm. The field of view (FOV) was 10-12 cm2, using an 256 × 192 imaging matrix and 2-4 acquisition averages. Figure 3 shows that tat-DOTA-Gd-labeled lymphocytes had significantly higher signal intensity compared to unlabeled cells, whose signal intensity was similar to that of agar. Conversely, the signal of tat-DOTA-Dy labeled cells was lower because of the predominant T2/T2* effect of the latter, which may be desirable for certain imaging studies. The R1 relaxivity of the gadolinium-labeled tatDOTA conjugate was 4.1 mM-1 s-1 in aqueous solution (20 °C, 1.5 T), similar to that reported for other Gd chelates (35). Tat-DOTA internalized in cells had a lower R1 relaxivity (2.2 mM-1 s-1) as predicted by theory of

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Figure 1. Synthesis. Tat-DOTA was labeled with either Gd, Dy, or 111In for cell uptake experiments. The typical intracellular distribution of tat peptide which is labeled with FITC is shown at the bottom right in a HeLa cell. Note the prominent intracellular and nuclear uptake.

limited water diffusion (35). Additional preliminary data indicates that cells other than murine lymphocytes (e.g., mouse neural progenitor cells C17.2; data not shown) can also be labeled by this approach. Our studies attaching paramagnetic labels to HIV-tat peptides have two major applications for in vivo MR imaging research. First, a variety of immune and other cells could be labeled ex vivo and cell trafficking could be studied noninvasively and repeatedly in recipient animals using MR imaging. Such cell tracking could be performed at high spatial resolutions (∼20 µm resolution)

and without significant image artifacts as frequently observed with superparamagnetic iron oxide labeled cells. Second, it now seems possible to synthesize membrane impermeable diagnostic probes and deliver them intercellularly through coupling to MTS peptides. Functionalized imaging reporters have recently been described that change their physical properties following interaction with a specific target including molecular beacons (36), molecular probes (37), and paramagnetic MR contrast agents (30, 31). The latter have been shown to alter their T1 relaxivity when conformational changes occur

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Figure 2. Uptake of tat-DOTA into mouse lymphocytes. One million murine lymphocytes were used to quantitate cellular uptake for each data point shown. Following incubation with tat-DOTA-111In, cells were washed extensively to remove any noninternalized compound. (A) Effect of increasing tat-DOTA-111In concentrations on cellular uptake (60 min incubation, 37°, top curve). Negligible uptake was observed with DOTA-111In at same condition (bottom curve). (B) Effect of time of incubation cellular uptake (at 40 nmol, 37 °C).

could have far reaching implications for both in vitro and in vivo applications of molecular imaging. ACKNOWLEDGMENT

The authors acknowledge the valuable help of Dr. U. Mahmood in performing the MR imaging experiments, and Dr. L. Josephson for many fruitful discussions and review of the manuscript. Supported by NIH Grants RO1 AI-CA46973 and R21DK55713. LITERATURE CITED

Figure 3. MR images of murine lymphocytes. Unlabeled (left) and labeled (right) murine lymphocytes (106 cells) were embedded as central pellets in agar cylinders and subjected to MR imaging [T1 weighted spin-echo (TR/TE) 300/11 for the tatDOTA-Gd compound, top; T2* weighted gradient echo (TR/TE/ angle) 50/20/20° for tat-DOTA-Dy, bottom]. As expected, Gd labeled cells appeared bright while Dy-labeled cells appeared dark, indicating that internalization of the paramagnetic labels occurred at sufficiently high quantities for MR imaging.

such as cleavage of galactose groups after galactosidase interaction (30) or interaction with calcium (31). These agents, as well as a myriad of other functional agents under development have potential applications for noninvasive in vivo imaging. However, to date one of the problems of some of these new reporters is their poor cell permeability and/or inability to attain intracellular concentrations required for imaging. The intracellular delivery of compounds designed to interact with specific intracellular targets by attachment to MTS peptides

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