Novel Tat-Peptide Chelates for Direct Transduction of Technetium

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Bioconjugate Chem. 2000, 11, 762−771

Novel Tat-Peptide Chelates for Direct Transduction of Technetium-99m and Rhenium into Human Cells for Imaging and Radiotherapy Valery Polyakov, Vijay Sharma, Julie L. Dahlheimer, Christina M. Pica, Gary D. Luker, and David Piwnica-Worms* Laboratory of Molecular Radiopharmacology, Department of Radiology and Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110. Received January 28, 2000; Revised Manuscript Received July 10, 2000

Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (-KGC) that provides an N3S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [99mTc]Tat-peptide species (4) that differed in retention time by ∼2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [99mTc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [99mTc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t1/2 < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 °C buffer compared to 37 °C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [99mTc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [99mTc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.

INTRODUCTION

Peptide-based imaging and radiotherapeutic agents offer high molecular target specificity and a great degree of flexibility in their design (1-4). Peptide-based imaging agents, exemplified by the commercially available agents Octreoscan and Acutech (5-7), often are composed of a domain for targeting cell-surface receptors and a moiety for stable chelation of desired radiometals (1, 2). Among these, technetium-99m (99mTc) is the most commonly used isotope in medical imaging (3). Readily available through 99 Mo/99mTc generator systems, 99mTc has an ideal photon energy for imaging applications and possesses a short half-life (6 h) compatible with the pharmacokinetics in vivo of many biological and clinical applications (1, 3, 8, 9). In addition, chelation chemistry for 99mTc has received much attention because these donor cores also enable radiotherapy using the chemically analogous 186Re/188Re species (10). Targeting of peptide-based imaging agents largely has been limited to extracellular or externally * To whom correspondence should be addressed. Phone: (314) 362-9356. Fax: (314) 362-0152. E-mail: piwnica-wormsd@ mir.wustl.edu.

oriented membrane receptors because charge, size, and pharmacokinetic considerations of conventional peptide constructs do not enable translocation across cellular membranes (2). The dielectric constant of the lipid bilayer of the plasma membrane poses a formidable barrier to the entry of charged compounds into cells (11). Because targeted delivery of peptide agents to intracellular compartments would enable a broad range of novel applications in medical imaging and radiotherapy, a peptide conjugate capable of incorporating medically relevant metals while permeating the plasma membrane is highly desired. Several transduction sequences capable of permeating the plasma membrane of cells and thereby enabling delivery of biomolecules and various “cargos” have been reported recently. These include the third helix of the homeodomain of Antennapedia (12), a peptide derived from the heavy chain variable region of an anti-DNA monoclonal antibody (13), and several viral proteins including Herpes simplex virus VP22 (14), HIV-1 Rev protein and HTLV-1 Rex protein basic domains (15), and HIV-1 Tat protein basic domain (16, 17). Although the exact mechanisms of membrane transduction are not well

10.1021/bc000008y CCC: $19.00 © 2000 American Chemical Society Published on Web 09/21/2000

Intracellular Transduction of Technetium Tat-Peptide Complexes

Bioconjugate Chem., Vol. 11, No. 6, 2000 763 Scheme 1

Figure 1. Structure of a [99mTc]Tat-peptide complex.

characterized, they generally have been described as achiral and independent of a receptor (12, 18). Membrane transduction peptides have been shown to deliver into cells a variety of polypeptides (14, 17, 19), immunoreactive epitopes (20), fluorescent probes (21), and derivatized superparamagnetic iron oxide nanoparticles (22). Herein we report a Tat-peptide basic motif for transduction of peptide chelates across plasma membranes as a delivery vehicle for imaging and radiotherapeutic isotopes. We designed prototypic peptides that constituted in their simplest form two functional domains, a membrane permeant peptide sequence derived from HIV-1 Tat protein residues 48-57 (GRKKRRQRRR) and a peptide-based chelator able to coordinate isotopes of metals useful in medical imaging and therapy (Figure 1). Novel radiolabeled Tat-peptide chelates are shown to rapidly and quantitatively concentrate by over 30-fold within the cytoplasm and nucleus of living human cells. EXPERIMENTAL PROCEDURES

Peptide Synthesis. Peptide conjugate 1 (Gly-Arg-LysLys-Arg-Arg-Gln-Arg-Arg-Arg-AHA--Lys-Gly-Cys) was prepared by solid-phase peptide synthesis using all L N-R-FMOC-protected amino acids and standard BOP/ HOBt coupling chemistry, except for the -Lys residue which used an N-R-tBOC, N--FMOC-Lys to direct peptide coupling to the -amine. AHA represents 6-aminohexanoic acid as an aliphatic linker between the Tat 4857 residues and the peptide chelating moiety. For peptides 2 and 3, the N-terminus Gly was omitted. Peptides were either N-terminus acetylated (1 and 2) or biotinylated (3), C-terminus amidated (1, 2, and 3), and deprotected by standard methods. Peptides were purified (>94%) by preparative C18 reversed-phase HPLC using as eluent 0.1% trifluoroacetic acid in water (0.1% TFA/H2O) modified with 0.1% trifluoroacetic acid in 90% acetonitrile/ 10% water [0.1% TFA/(90% CH3CN/H2O)] by a linear gradient (5-40% over 40 min) flowing at 1.0 mL/min. A single HPLC peak was observed for each peptide conjugate. The identity of peptide conjugates 1-3 was confirmed by amino acid analysis [1, 14 amino acids (Gln 1; Gly 2; Cys 1; Lys 3, Arg 6, AHA 1); 2 and 3, 13 amino acids (Gln 1; Gly 1; Cys 1; Lys 3; Arg 6, AHA 1] and electrospray mass spectrometry (1, m/z 1839.0, calcd, C74H143N37O16S1, 1839.3; 2, m/z 1782.8, calcd C72H140N36O15S1, 1782.2; 3, m/z 1965.6, calcd C80H151N38O16S2, 1965.5). Preparation of Radiolabeled [99mTcvO]Tat-Peptide. Tat-peptide conjugates were labeled with 99mTc by ligand exchange using [99mTc]glucoheptonate as the ligand exchange reagent (Scheme 1) (7). A commercially available stannous glucoheptonate radiopharmaceutical kit [Sn(II)Cl2‚2H2O, 0.14 mg; sodium glucoheptonate, 200 mg; Glucoscan, DuPont Pharma, Billerica, MA] was reconstituted with 1.0 mL of [99mTc]Na(TcO4) (50 mCi) in isotonic saline obtained by eluting a commercial radionuclide 99Mo/99mTc generator and allowed to stand for 15 min at room temperature. In a small glass vial, to a 10 µL aliquot of Tat-peptide conjugate stock solution

(10 mg dissolved in 1 mL of 0.9% saline or water), [99mTc]glucoheptonate solution (15-50 µL), and sufficient water to generate a final volume of 100 µL were added, and the reaction was allowed to proceed at room temperature for 15 min. Radiochemical yields (>95%) of oxotechnetium [99mTc]Tat-peptide complexes were determined by TLC using silica gel developed with either 15% TFA or H2O as indicated and scanning radiometric detection (Bioscan, Inc., Washington DC). By TLC with TFA, [99mTc]Tat complexes 4, 5, and 6 from peptides 1, 2, and 3, respectively, each showed an Rf ≈ 0.45, readily distinguished from [99mTc]glucoheptonate (Rf ) 0.95) and [99mTc]TcO4- (Rf ) 0.95). By TLC with H2O, [99mTc]Tat complexes remained at the origin while [99mTc]glucoheptonate and [99mTc]TcO4- moved with the solvent front. Radiochemical purity (>90%) was determined by radiometric RP-HPLC using the solvent gradient system described above as well as a linear gradient (0-60% over 60 min) also flowing at 1.0 mL/min. Specific activity was estimated to be 2 × 108 mCi/mol. Preparation of [RevO]Tat-Peptide. [Re]Tat-peptide conjugates also were obtained through ligand exchange using [Re]glucoheptonate as the ligand exchange reagent (7). To 0.1 mL of a freshly prepared stock solution [sodium R-D-glucoheptonate, 200 mg, 0.81 mmol and tin(II)chloride dihydrate, 18.4 mg, 0.082 mmol in 1 mL of distilled water] was added 0.1 mL of a stock solution (ammonium perrhenate, 14.9 mg, 0.055 mmol in 1 mL of water), and the mixture allowed to stand for 15 min at room temp. To the mixture was added the appropriate Tat-peptide conjugate (1 mg, ∼0.4 µmol) in water, and the reaction allowed to proceed at room temperature for 30 min. RP-HPLC analysis was performed using the solvent gradient system described above and the desired fractions collected. Identity of isolated [Re]Tat-peptide complexes was confirmed by electrospray mass spectrometry (acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-ArgAHA--Lys-Gly-Cys[Re] (7), m/z 2039.0, calcd C74H140N37O17Re1S1, 2038.4). Preparation of Tat-Peptide-Fluorescein. Tatpeptide conjugates were labeled on the C-terminal thiol with fluorescein maleimide (FM) as described previously (21). Briefly, in a small glass vial, Tat-peptide conjugate (1 mg) was dissolved in phosphate buffered saline (PBS, pH 7.4) and reacted with 1.2 eq of fluorescein maleimide dissolved in dimethylformamide for 2 h in the dark at room temperature. Fluorescent peptide was purified by RP-HPLC (single peak; purity >97%) using the above

764 Bioconjugate Chem., Vol. 11, No. 6, 2000

gradient conditions and lyophilized in the dark. Identity of the desired fluorescein tagged Tat-peptide was confirmed by electrospray mass spectrometry (acetyl-GlyArg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-AHA--Lys-GlyCys-FM (8), m/z 2266.0, calcd C98H156N38O23S1, 2266.6; acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-AHA--LysGly-Cys-FM (9), m/z 2210.0, calcd C96H153N37O22S1, 2209.6). Cell Culture. Monolayers of human epidermoid carcinoma KB 3-1 cells and the colchicine-selected KB 8-5 derivative cell lines were grown at 37 °C in an atmosphere of 5% CO2 as previously described (23, 24). For experiments, cells were plated in 100-mm Petri dishes containing seven 25-mm glass coverslips on the bottom and grown to subconfluence in DMEM (GIBCO, Grand Island, NY) supplemented with L-glutamine (1%), penicillin/streptomycin (0.1%), and heat-inactivated fetal calf serum (10%) in the absence or presence of 10 ng/mL colchicine, respectively. Human Jurkat leukemia cells were maintained in RPMI also supplemented with L-glutamine, penicillin/ streptomycin, and 10% fetal calf serum at 37 °C in an atmosphere of 5% CO2 (25). Cellular Uptake and Washout Studies of [99mTc]Tat-Peptide Conjugates. Control solution for tracer uptake experiments was a modified Earle’s balanced salt solution (MEBSS) containing (mM): 145 Na+, 5.4 K+, 1.2 Ca2+, 0.8 Mg2+, 152 Cl-, 0.8 H2PO4-, 0.8 SO42-, 5.6 dextrose, 4.0 HEPES, and 1% bovine calf serum (vol/vol), pH 7.4 ( 0.05. A 130 mM K+/20 mM Cl- solution was made by equimolar substitution of potassium methanesulfonate for NaCl as described (26). Kinetic experiments of [99mTc]Tat-peptide complexes were performed in Jurkat leukemia cells suspended in MEBSS with minor modifications of methods described in the literature (27). Transport experiments were performed in siliconized microfuge tubes and initiated by addition of 732.5 µL cells at 5 × 106 cells/ml to 10 µL of MEBSS containing [99mTc]Tat-peptide complex and 7.5 µL of vehicle alone or of any added drug in vehicle at 100-fold the desired concentration. Unless stated otherwise, [99mTc]Tat-peptide complex was added to MEBSS accompanied by molar excess unlabeled Tat-peptide as obtained directly from the labeling procedure; final total peptide concentration was typically 0.5-1 µM (50 nmol/ mCi; 10 µCi/mL). Tubes were incubated in a 37 °C water bath with occasional mixing. The reaction was terminated by spinning 250 µL aliquots from the reaction for 10 s through 800 µL of a 75:25 mixture of silicon oil (density ) 1.050; Aldrich) and mineral oil (density ) 0.875; Acros). An aliquot of the aqueous phase was obtained to normalize extracellular concentration of the Tc-peptide to cell-associated activity, then the oil and aqueous phases were aspirated and the cell pellet extracted in 0.5 mL of 1% SDS, 10 mM sodium borate. For peptide washout experiments, cells were first incubated to plateau uptake (10 min) in MEBSS loading buffer (37 °C), collected by rapid centrifugation and the pellet resuspended in 750 µL peptide-free MEBSS (4 °C) to clear extracellular tracer. Following another rapid spin, the cell pellet was resuspended in peptide-free MEBSS (37, 25, or 4 °C) for various times and the experiment terminated as above. All cell extracts were assayed for protein by BCA analysis (Pierce Chemical Co.) using bovine serum albumin as the protein standard. Cell extracts, stock solutions, and extracellular buffer samples were assayed for gamma activity in a well-type sodium iodide gamma counter (Cobra II, Beckman; 130-165 keV window). Absolute concentration of total [Tc]Tat-peptide complex in solution was determined from the specific

Polyakov et al.

activity of Tc, based on equations of Mo/Tc generator equilibrium (28). Transport data are reported as pmol of peptidei (mg protein)-1 (µMo)-1, wherein peptidei represents total peptide within the cells and (µMo)-1 represents concentration of total peptide in the extracellular buffer (29). Data are generally reported as mean ( SEM. Fluorescence Microscopy. Exponentially growing Jurkat cells were incubated in fresh media or MEBSS containing the fluorescein labeled Tat-peptide conjugate (0.5-1 µM) at 37 or 4 °C for 10-20 min. Cells were then cytospun onto glass slides, fixed at room temperature or on ice by direct addition of 4% (v/v) paraformaldehyde in PBS to the adherent cells for 10 min and then rinsed three times with PBS (1 min each). Similarly, human KB 3-1 and KB 8-5 epidermoid carcinoma cells growing on coverslips or chamber slides were incubated in MEBSS containing the fluorescein labeled Tat-peptide conjugate at 37 °C for 10 min, then fixed in 4% paraformaldehyde at room temperature and rinsed three times with PBS. Cells were then mounted with anti-fading mounting medium following the recommended procedures of the manufacturer (Vector). Distribution of fluorescein conjugates was analyzed on a Zeiss epifluorescence microscope or laser-scanning confocal fluorescence imaging system coupled to a Nikon microscope equipped with a CCD interfaced to a PC (30). Stability of Radiolabeled Peptide in Vitro and in Vivo. The stability in vitro of [99mTc]Tat-peptide 4 in aqueous buffers between pH 4 and 9 was evaluated by incubation of radiolabeled peptide (10 mg/mL) for 1 h at RT in HEPES (10 mM) or Tris base (50 mM) buffer pH adjusted with HCl or NaOH, followed by TLC and scanning radiometric analysis. Additional effects of reduced glutathione (2.5 mM), cysteine (10 µM and 2.5 mM) or EDTA (2.5 mM) on radiolabeled Tat-peptide stability was performed by addition of competing chelator to buffers at each pH. Stability in vitro was further assessed by radio-TLC after incubation of [99mTc]Tat-peptide for 1 h at 37 °C in human serum, bovine calf serum, murine urine, culture media containing 10% fetal calf serum as well as MEBSS transport buffer. In addition, human Jurkat cells were incubated with [99mTc]Tat-peptide 4 in MEBSS transport buffer for 1 h at 37 °C, centrifuged, and the cell pellet lysed in modified mammalian lysis buffer {100 mM NaCl; 50 mM Tris base (pH 8); 2 mM dithiothreitol; 0.5% NP-40; 1 mM phenylmethylsulfonylfluoride; 10 µg/mL leupeptin; 10 µg/mL aprotinin}. Radio-TLC analysis of both extracellular buffer and cell lysate was performed. Furthermore, stability in vivo of radiolabeled Tat-peptide was characterized by radio-TLC analysis of serum and urine samples 30 min after intravenous administration of 270 µCi of [99mTc]Tat-peptide 4 to FVB mice. Biodistribution and Imaging Studies. Vertebral animal procedures were approved by the appropriate institutional review committee. Distribution of [99mTc]Tat-peptide complex 4 in tissues of Balb/c mice was determined as previously described (31, 32). Mice were anesthetized by metofane inhalation and injected with 4 (2 µCi in 50 µL saline) via bolus injection through a tail vein. Animals were sacrificed by cervical dislocation at 5, 30, 60, and 120 min postinjection (n ) 3 each). Blood samples were obtained by cardiac puncture and tissues harvested rapidly. Gamma activity in organ samples was counted for 1 min, or until two standard deviations of sampling were below 0.5%. Data are expressed as percent of injected dose (% id) per organ [(organ µCi) (injected µCi)-1 × 100] or % id per gram of tissue [(tissue µCi) (injected µCi)-1 (g tissue)-1 × 100].


Figure 2. Radiometric (top) and UV (bottom) RP-HPLC chromatograms of [99mTc]Tat-peptide complex 4 and [Re]Tatpeptide complex 7, respectively. Isolation and mass spectrometry of each Re species revealed identical masses (m/z 2039.0) consistent with formation of syn and anti isomers upon chelation of the metal.

For imaging studies, Balb/c mice also were lightly anesthetized with metofane inhalation. [99mTc]Tat-peptide complex 4 (200 µCi in 50 µL saline) was injected via a tail vein into mice positioned under a gamma scintillation camera (Siemens Basicam, Siemens Medical Systems, Iselin, NJ; 5 mm pinhole collimator; 20% energy window centered over 140 keV photopeak of Tc-99m). Sequential posterior images of mice were collected for 3 min each at 1, 30, and 60 min postinjection of the radiotracer with the mouse recovering and being reanesthetized just prior to each timepoint. A 256 × 256 image matrix with correction for radioactive decay was used on a PC platform and standard image analysis software. No corrections were made for scatter or attenuation. Whole body distribution of the complexes are presented with gray scale images. RESULTS

Chemistry. Tat-peptide conjugates were synthesized by standard solid-phase methods and their corresponding 99mTc- and Re-complexes were obtained by ligand exchange using [99mTc]glucoheptonate or [Re]glucoheptonate, respectively (Scheme 1). Incorporation of a metal into the N3S donor core has been shown to result in the formation of three five-membered rings and create a chiral center (33). Such structures typically resemble a square pyramid with the nitrogens at the base and the oxygen in the apical position (33) and are known to be stable (34). Radiochemical yield (>95%) and purity (>90%) of the oxotechnetium complexes were determined by TLC using silica gel with scanning radiometric detection and by radiometric RP-HPLC, respectively. By TLC, [99mTc]Tat peptide complexes were readily distinguished from [99mTc]glucoheptonate and [99mTc]TcO4- which moved at or near the solvent front. RP-HPLC analysis and radiometric detection of [99mTc]Tat-peptide 4 revealed two closely eluting peaks (Figure 2). Similarly, RP-HPLC analysis and UV detection of [Re]Tat-peptide 7 also revealed two eluting peaks with retention times differing by ∼2 min (Figure 2), close to the radiometric peaks observed with the 99mTc-labeled species. Furthermore, a second gradient system revealed similar results. Each Re peak was isolated and the identity of the oxorhenium Tat-peptide complex confirmed by mass spectrometry. Because both peaks had identical mass (m/z 2039.0 each; calcd 2038.4), we concluded that each represented an

Bioconjugate Chem., Vol. 11, No. 6, 2000 765

Figure 3. Effect of pH and competing chelation compounds on stability of radiolabeled Tat-peptide. Following incubation of [99mTc]Tat-peptide complex 4 for 1 h at RT in HEPES or Tris buffer adjusted to the indicated pH in the absence (b) or presence of reduced glutathione [2.5 mM (9)], cysteine [10 µM (2)], cysteine [2.5 mM (1)], or EDTA [2.5 mM (()], percent intact complex was determined by radio-TLC.

isomer of the N3S chelation moiety in relation to the metal-oxygen bond due to participation of the chiral R-C atom of lysine in the chelation ring (2, 33, 35). The data were consistent with formation of the two anticipated diastereoisomers, the apical oxygen being syn and anti relative to the side chain of the Lys residue (see the asterisk in Scheme 1), as validated with analogous N3S structures. Separation of the isomers of Re-Tat-peptide complex 7 was accomplished by RP-HPLC and the kinetics of interconversion studied in aqueous solution at RT. For analysis, an HPLC fraction (assigned to isomer a) was collected, lyophilized, resuspended in water or PBS and monitored over 48 h. Interconversion of isomer a was not observed in deionized water. However, in PBS (pH 7.4), the emergence of isomer b was evident after 1 h at RT and a peak ratio of ∼2:1 (isomer a:isomer b) was observed at 2 h. After 48 h, the ratio remained ∼2:1, the inverse of the original mixture of 7 (see Figure 2). Thus, while PBS facilitated interconversion of the chelate, equilibrium was not observed with the peptide conjugate over this time interval. A proposed mechanism for interconversion of an analogous isolated N3S chelate involves the coordination of a water molecule in the sixth position on the metal, trans to the metal-oxo bond (33). To study the stability of the Tat-peptide chelate conjugates in vitro, radio-TLC analysis of [99mTc]Tatpeptide 4 was examined after incubation of peptide in buffered solution ranging in pH from 4 to 9 (Figure 3). As shown, [99mTc]Tat-peptide 4 was very stable to transchelation between pH 4-9, a physiologically relevant range of pH values likely to be encountered in vivo. Percent intact complex remained >97% over 1 h. The presence of reduced glutathione (2.5 mM; ∼100000-fold molar excess over labeled peptide complex), a competing physiologic chelator with modest binding affinity, showed no effect. Furthermore, [99mTc]Tat-peptide 4 remained >96% intact in the presence of physiological concentrations of the strong chelator cysteine (10 µM; ∼1000-fold molar excess), even under alkaline conditions. However, under extreme conditions of highly concentrated cysteine (2.5 mM) or EDTA (2.5 mM), evidence of over 50% transchelation was observed at pH g7. Additionally,


Figure 4. Kinetics of [99mTc]Tat-peptide complex 4 accumulation into human Jurkat cells. Cells were incubated in MEBSS loading buffer for the indicated times, washed, and cellassociated activity determined. Each point represents the mean of three to four determinations; bars represent ( SEM when larger than the symbol. Plot is representative of four independent experiments. (Inset) Concentration dependence of plateau accumulation of 4. Twenty minute net uptake values at the indicated extracellular concentrations of 4 are plotted.

incubation of [99mTc]Tat-peptide in human serum, bovine serum, cell culture media or MEBSS transport buffer for 1 h at 37 °C showed no evidence of metabolites or transchelation reactions by TLC (H2O) (data not shown), further suggesting that the [99mTc]Tat-peptide was resistant to the action of serum proteases and natural chelators. Cellular Transport Kinetics of [99mTc]Tat-Peptides. We first examined the overall uptake kinetics of [99mTc]Tat-peptide 4 in human Jurkat leukemia cells. Jurkat cells rapidly accumulated 4, approaching a plateau in less than 5 min (Figure 4). This exceeded by an order of magnitude the uptake kinetics previously reported for an organometallic hydrophobic cation, [99mTc]Sestamibi, which also is known to localize intracellularly (29, 36). Steady-state values for the [99mTc]Tat-peptide complex were 116 ( 3 pmol (mg protein)-1 (µMo)-1 (n ) 4). Given a typical cell water space of 4 µL (mg protein)-1 (23), this would indicate an intracellular/extracellular ratio for complex 4 of ∼30, consistent with the complex being rapidly concentrated within cells. Furthermore, TLC analysis of cell lysates showed >95% intact complex (data not shown). To further characterize transport of the complex, plateau accumulation of [99mTc]Tat-peptide 4 in Jurkat cells (10 min incubation) was determined as a function of extracellular concentration of the agent by addition of increasing amounts of radiolabeled peptide to the buffer (at a fixed specific activity). While readily detectable at concentrations as low as 7 nM, cell content of 4 rose in proportion to the extracellular concentration of the complex. There was evidence of concentration saturation as extracellular concentrations rose into the 8 µM range (Figure 4, inset). The shape of the concentrationdependence curve implied the existence of a diffusive component and a saturable binding component to the overall uptake mechanism (31). Curve fitting of the data (37) suggested a half-maximal value of 2 µM for the saturable component. Washout of [99mTc]Tat-peptide complex 4 also showed very rapid kinetics. Washout was ∼90% complete within

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Figure 5. Washout kinetics of [99mTc]Tat-peptide complex 4 from human Jurkat cells at 37 °C. Cells were incubated (10 min) in MEBSS loading buffer (37 °C), washed, and then incubated in peptide-free MEBSS (37 °C). Cell-associated activity as a function of time in washout buffer is shown. Each point represents the mean of three to four determinations; bars represent ( SEM when larger than the symbol. Plot is representative of three independent experiments. (Inset) Concentration dependence of cellular residual activity of 4. Sixty minute washout residual values after cell loading at the indicated extracellular concentrations of 4 are plotted.

20 min (Figure 5). This may have favorable impact on signal-to-noise for nontarget tissues regarding use of this general class of agents in vivo. However, a residual compartment of slowly exchanging or retained activity was observable for longer time points. This residual was a function of extracellular peptide concentration (Figure 5, inset). Because the Arg-rich Tat protein basic domain is known to bind to viral RNA, this residual compartment may represent binding to analogous nuclear components in human cells, but this remains to be ascertained. The radiosynthetic reaction for the Tat-peptide complexes inherently yields a mixture of isomeric [99mTc]Tatpeptide complexes (as described above) as well as ∼10000fold molar excess unlabeled Tat-peptide conjugate. The latter arises as a consequence of the extremely high specific activity of the radiolabeling isotope 99mTc (3). To determine whether human cells could distinguish between putative syn and anti isomers of purified Tatpeptide complexes, fractions containing pure isomers of [99mTc]Tat-peptide complex 4 were isolated by RP-HPLC, lyophilized and each added to MEBSS buffer for accumulation experiments into Jurkat cells (10 min incubation, a short time compared to the solution interconversion times shown above). In Jurkat cells, the 10-min uptake of purified [99mTc]Tat-peptide isomer b was ∼60% of the uptake of isomer a (Table 1). However, in the same cells, when 20000-fold molar excess unlabeled Tatpeptide conjugate was added to purified [99mTc]Tatpeptide isomers in buffer, thereby simulating standard peptide mixtures, 10-min uptake of isomer b was now ∼80% of the uptake of isomer a. In addition, no consistent stereospecific trend was observed for the washout residual (Table 1). Thus, while net accumulation of putative syn and anti isomers of [99mTc]Tat-peptide complex 4 were not exactly equal, conformation per se produced only modest effects on the net cell uptake and washout of these [99mTc]Tat-peptide complexes. Furthermore, the 3-4-fold enhancement of cellular accumulation of purified isomers of [99mTc]Tat-peptide in the presence of



Table 1. Uptake and Washout of Isomers of [99mTc]Tat-peptide 4 in Human Jurkat Cellsa peptide

isomer a

10-min net uptake [pmol (mg protein)-1 (µMo)-1] purified [99mTc]Tat-peptide 62.8 ( 4.1 added unlabeled peptide 185.9 ( 10.9 30-min washout residual [pmol (mg protein)-1 (µMo)-1] 99m purified [ Tc]Tat-peptide 10.8 ( 1.1 added unlabeled peptide 11.2 ( 0.8

isomer b

36.6 ( 5.2 149.2 ( 4.5

7.9 ( 1.4 23.8 ( 4.8

a Cells were incubated in MEBSS loading buffer (10 min; 37 °C) containing HPLC-purified isomers of 4 in the absence or presence of ∼20000-fold molar excess unlabeled peptide, and then analyzed, or washed and then incubated in peptide-free MEBSS (30 min; 37 °C) prior to analysis. Cellular content of 4 is expressed as [pmol (mg protein)-1 (µMo)-1]. Values represent the mean ( SEM of three to four determinations each.

Table 2. Effect of Various Drugs on Net Uptake of [99mTc]Tat-peptide 4 in Human Jurkat Cells drug

10-min net uptake [pmol (mg protein)-1 (µMo)-1]

control MEBSS 0.1% DMSO vehicle Colchicine (100 ng/mL) Taxol (1 µM) Nocodozole (5 µg/mL) Cytochalasin D (1 µM) Brefeldin A (2.5 µg/mL) Wortmannin (100 nM)

79.1 ( 2.0 94.4 ( 20.2 96.5 ( 11.8 72.0 ( 4.3 66.8 ( 1.7 76.2 ( 1.9 79.4 ( 9.8 82.9 ( 10.0

Figure 6. Effect of temperature on accumulation of [99mTc]Tat-peptide complex 4 into human Jurkat cells. Cells were incubated in MEBSS loading buffer at 37 °C (b) or 4 °C (O) for the indicated times, washed, and cell-associated activity determined. Each point represents the mean of four determinations, except the 30 min point at 4 °C (n ) 1); bars represent ( SEM when larger than the symbol. Plot is representative of two independent experiments.

a Cells were incubated in MEBSS loading buffer (10 min; 37 °C) containing 4 and the indicated drugs prior to analysis. See the legend of Table 1 for details.

20000-fold molar excess unlabeled peptide (Table 1) obviates a simple receptor-ligand interaction for the net cell uptake process on one hand and simple diffusion on the other hand. These data provide evidence for a translocation mechanism with overall high capacity and a modest degree of positive cooperativity. Because several membrane permeant peptides have been reported to accumulate within cells by mechanisms related to cytoskeletal function (14), several inhibitors known to impact microtubules, actin filaments, and various cytoskeletal-mediated vesicular transport pathways were tested. In Jurkat cells, neither the microtubule-disrupting agents colchicine (100 ng/mL), taxol (1 µM), nocodozole (5 µg/mL), the filament-disrupting agent cytochalasin D (1 µM), the trans-Golgi transport inhibitor brefeldin A (2.5 µg/mL), nor the phosphatidylinositol-3 kinase inhibitor wortmannin (100 nM) had any significant effect on net cell uptake of [99mTc]Tat-peptide 4 (Table 2). Thus, the pathway for accumulation of [99mTc]Tat-peptide was unlikely to be mediated by a microtubulin or cytoskeletal-mediated transport mechanism. Furthermore, ice-cold buffer (4 °C) not only failed to inhibit uptake, but modestly enhanced net accumulation of the complex over time (Figure 6). In addition, after loading in 37 °C buffer, washout into ice-cold buffer was only modestly inhibitory (Figure 7). These data point to an unique cell membrane translocation pathway not highly dependent on endocytosis, vesicular trafficking or cellular metabolism. To further examine potential mechanisms of localization, Jurkat cells were incubated with standard [99mTc]Tat-peptide 4 in control MEBSS or buffer comprised of 130 mM K+/20 mM Cl- plus valinomycin (1 µg/mL), a potassium ionophore (29). When cells are incubated in this high K+ buffer, electrical potentials of the mitochon-

Figure 7. Effect of temperature on washout of [99mTc]Tatpeptide complex 4 from Jurkat cells. Cells were incubated (10 min) in MEBSS loading buffer (37 °C), washed, and then incubated in peptide-free MEBSS at 37 °C (b), 25 °C (1), or 4 °C (9). Cell-associated activity as a function of time in washout buffer is shown. Each point represents the mean of three to four determinations; bars represent ( SEM when larger than the symbol.

drial membrane (∆Ψ), and plasma membrane (Em) are depolarized toward zero, eliminating the inward driving force for uptake of cationic molecules (38). While Tatpeptide has amphiphilic cationic properties (21), net uptake of [99mTc]Tat-peptide complex 4 under isoelectric conditions was actually 42 ( 3% greater than control (data not shown), rather than decreased, indicating that the mechanism of Tat-peptide accumulation was independent of membrane potential. The effect of N-terminus modifications on cellular uptake of the [99mTc]Tat-peptide conjugates was also explored. Deletion of the N-terminus Gly as in [99mTc]Tat-peptide 5 modestly reduced both Tat-peptide net uptake and the washout residual in Jurkat cells (Table 3), but resulted in an increased net uptake/washout ratio.


Polyakov et al.

Table 3. Effect of N-Terminus Modification on Net Uptake and Washout of [99mTc]Tat-peptides in Human Jurkat Cells [pmol (mg protein)-1 (µMo)-1] peptide

20-min net uptake

30-min washout residual

[99mTc]Tat-peptide 4 [99mTc]Tat-peptide 5 [99mTc]Tat-peptide 6

77.9 ( 3.5 61.7 ( 6.1 42.7 ( 3.2

24.3 ( 7.6 11.0 ( 1.8 24.3 ( 6.3

a Cells were incubated in MEBSS loading buffer (20 min; 37 °C) containing 4, 5, or 6, and then analyzed, or washed and then incubated in peptide-free MEBSS (30 min; 37 °C). Cell content of peptide was determined as indicated. See the legend of Table 1 for details.

Figure 8. Cellular accumulation of fluorescein labeled Tatpeptide conjugate (8) in human Jurkat and KB-3-1 tumor cells. Cells were incubated with substrate for 10-20 min at 37 or 4 °C as indicated followed by fixation. Panels show fluorescence micrographs of Jurkat cells incubated at 37 °C in buffer containing fluorescein maleimide (1 µM) alone (A), at 37 °C in buffer containing 8 (B), and at 4 °C in buffer containing 8 (C) as well as a confocal micrograph of KB-3-1 cells incubated at 37 °C in buffer containing 8 (D). Note green fluorescence from labeled peptide residing in both cytosolic and nuclear (putative nucleolar) components. The cytosolic compartment containing fluorescent peptide, while more obvious in KB-3-1 tumors cells, appears as a rim surrounding the Jurkat cell nuclei, typical of lymphocytic cell types. Panel D, bar ) 5 µm; panels A-C, magnified ∼2× panel D.

However, N-terminus biotinylation of the shortened peptide (6) decreased net uptake while increasing the washout residual resulting in a lower uptake/washout ratio (Table 3). Fluorescence Microscopy. While the tracer experiments advantageously determined cell-associated radioactivity in a quantitative manner, the exact subcellular location of [99mTc]Tat-peptides cannot be evaluated by population-based γ-counting techniques. To directly localize and thereby prove intracellular distribution of the Tat-peptide chelate construct, uptake experiments were performed with a fluorescein derivatized Tat-peptide conjugate (8) using suspension cultures of human Jurkat cells. As expected, cells did not show internalization of fluorescein maleimide when the nonconjugated fluorophore was added to the media or MEBSS alone (Figure 8A). However, fluorescence microscopy revealed diffuse cytoplasmic and focal nuclear accumulation of the fluo-

Figure 9. Scintigraphic gray scale images of [99mTc]Tatpeptide complex 4 in a Balb/c mouse. Following bolus intravenous injection of 4, posterior images of the mouse were obtained with a gamma scintillation camera. Representative planar images obtained 3 min (A), 30 min (B), and 60 min (C) postinjection are shown. Rapid whole body distribution is followed by predominantly renal clearance of the [99mTc]Tatpeptide complex and appearance in the urinary bladder. Arrows demarcate liver (liv), kidneys (k), and bladder (bl).

rescein derivatized Tat-peptide in all Jurkat cells (Figure 8B). Overall, the multifocal nuclear staining pattern was suggestive of nucleolar localization of the conjugate. Interestingly, the same uptake and localization pattern was observed at 4 °C (Figure 8C). In both KB 3-1 epidermoid carcinoma cells (Figure 8D) and MDR1 P-glycoprotein-expressing multidrug resistant KB 8-5 cells (data not shown), a cytoplasmic and focal nuclear accumulation pattern also was observed, providing evidence for the ubiquitous nature of tumor cell uptake for this Tat-peptide conjugate. The results in KB 8-5 cells also suggested that P-glycoprotein, an efflux pump of many hydrophobic cationic compounds (39), was not involved in the cell permeation pathway of Tat-peptides. Peptide 9 also showed a similar subcellular localization pattern (data not shown). Overall, the fluorescence staining pattern provided direct evidence for the intracellular localization of these novel Tat-peptide chelate based imaging and radiotherapeutic agents. Whole Body Imaging and Biodistribution Analysis. Exploiting the 140 keV emission photon of 99mTc, preliminary imaging of Balb/c mice was performed by gamma scintigraphy following tail vein bolus injection of [99mTc]Tat-peptide complex 4 (200 µCi). The complex was observed to rapidly distribute throughout the body, in a pattern initially consistent with organ perfusion (Figure 9). Of note, dynamic analysis of serial images as well as biodistribution data (Table 4) revealed that the complex reached peak organ levels within the first few minutes after injection and displayed modestly rapid blood clearance, favorable pharmacokinetic properties for potential uses of these complexes as imaging agents. Over the subsequent 2 h, [99mTc]Tat-peptide complex 4 was cleared by both renal and hepatobiliary excretion with activity appearing in the urinary bladder and bowel (Figure 9 and Table 4). Analysis of [99mTc]Tat-peptide stability in vivo also was performed. In FVB mice, radio-TLC analysis of serum and urine samples 30 min after intravenous injection of [99mTc]Tat-peptide 4 revealed >95% intact complex in serum, but only 65% intact complex in the urine. However, direct addition of [99mTc]Tat-peptide 4 to murine urine samples ex vivo for 1 h at 37 °C showed no evidence of transchelation, thus localizing the observed transmetalation/metabolic reactions to the renal parenchyma in situ.



Table 4. Biodistribution of [99mTc]Tat-peptide 4 in Balb/c Mice 5 min

a

blood lung liver spleen kidney muscle fat heart brain bone small intestine large intestine skin

12.22 ( 0.78 1.95 ( 0.03 23.73 ( 1.47 1.21 ( 0.01 13.55 ( 0.68 17.08 ( 0.88 2.89 ( 0.40 0.82 ( 0.05 0.13 ( 0.01 14.19 ( 0.54 8.16 ( 0.36 3.42 ( 0.28 8.48 ( 0.59

blood lung liver spleen kidney muscle fat heart brain bone small intestine large intestine skin

10.81 ( 0.92 17.13 ( 0.79 22.65 ( 1.47 18.81 ( 1.22 49.53 ( 4.06 3.02 ( 0.19 3.06 ( 0.48 9.42 ( 0.78 0.39 ( 0.05 5.03 ( 0.40 6.77 ( 0.38 4.49 ( 0.42 4.47 ( 0.11

30 min % id/organ 4.67 ( 0.13 0.89 ( 0.03 30.15 ( 0.96 0.45 ( 0.02 10.57 ( 1.00 9.97 ( 1.09 1.20 ( 0.18 0.20 ( 0.01 0.06 ( 0.00 10.69 ( 0.51 5.01 ( 0.41 0.89 ( 0.08 9.41 ( 0.18 % id/gram 4.07 ( 0.01 7.66 ( 0.37 27.64 ( 0.57 7.20 ( 0.43 36.94 ( 4.08 1.75 ( 0.23 1.25 ( 0.17 2.17 ( 0.12 0.18 ( 0.01 3.73 ( 0.23 4.73 ( 0.27 1.26 ( 0.15 4.92 ( 0.08

60 min

120 min

2.82 ( 0.17 0.43 ( 0.06 26.05 ( 0.67 0.36 ( 0.01 6.67 ( 0.38 9.00 ( 3.44 0.67 ( 0.14 0.10 ( 0.01 0.04 ( 0.01 7.24 ( 0.24 10.74 ( 0.70 0.55 ( 0.01 5.10 ( 0.29

1.58 ( 0.05 0.29 ( 0.03 18.36 ( 0.33 0.33 ( 0.00 4.61 ( 0.01 2.42 ( 0.41 0.37 ( 0.03 0.05 ( 0.00 0.03 ( 0.00 4.98 ( 0.08 17.12 ( 0.87 1.84 ( 0.19 1.88 ( 0.09

2.62 ( 0.24 4.09 ( 0.77 26.60 ( 1.70 5.48 ( 0.52 25.08 ( 1.60 1.62 ( 0.56 0.73 ( 0.13 1.09 ( 0.11 0.11 ( 0.02 2.69 ( 0.18 10.13 ( 0.94 0.85 ( 0.06 2.83 ( 0.17

1.38 ( 0.07 2.60 ( 0.23 17.25 ( 0.48 4.88 ( 0.19 16.99 ( 0.53 0.42 ( 0.08 0.38 ( 0.03 0.59 ( 0.03 0.08 ( 0.01 1.73 ( 0.04 15.50 ( 0.67 2.82 ( 0.30 0.98 ( 0.05

Mean ( SEM of three determinations at each time point. Error values of 0.00 indicate

Novel Tat-Peptide Chelates for Direct Transduction of Technetium

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