Novel Tat-Peptide Chelates for Direct Transduction of Technetium-99m and Rhenium into Human Cells for Imaging and Radiotherapy

Abstract

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 (epsilon-KGC) that provides an N(3)S 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 [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 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. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees 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 [(99m)Tc]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 [(99m)Tc]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.

Translocation of Analogues of the Antimicrobial Peptides Magainin and Buforin across Human Cell Membranes

“…5). Similar behaviors have been reported for a rhodaminelabeled Tat-(48 -60) peptide (38) and a 99m Tc-chelated Tat peptide (39).…”

“…4) by HeLa cells. In contrast, the kinetics of 99m Tcchelated Tat peptide internalization into Jurkat cells was largely changed by a lowering of the temperature (39). The uptake of Tat-(48 -60) (38) and 99m Tc-chelated Tat (39) by HeLa and Jurkat cells reached a maximum at 60 -90 min and 5 min, respectively, whereas that of Tat-(47-57) by HeLa cells continued to increase at least up to 120 min (Fig.…”

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

“…For this probe, a modified Tat peptide sequence has been used as the targeting moiety. A number of studies using Tat-derived peptides have demonstrated transport and intracellular delivery of molecular imaging as well as therapeutic agents into various cell types and tissues (19,36,(39)(40)(41)(42). In addition, we have previously shown the utility of this peptide for enhancing the intracellular accumulation of a conjugated fluorophore in relevant target cells, in this case, RGCs (20).…”

Radiolabeling of MAG3-morpholino oligomers with 188Re at high labeling efficiency and specific radioactivity for tumor pretargeting

“…In the case of carriers that can withstand harsh environments, post-conjugation labeling should be possible and more practical. However, although a few post-conjugation 188 Re labeling have been reported (Guhlke et al, 1998;Pearson et al, 1996;Zinn et al, 2000;Polyakov et al, 2000;Gestin et al, 2001), to our knowledge, none describes 188 Re labeling of a biomolecule at high labeling efficiency and high specific radioactivity. This laboratory has been using S-acetyl protected and N-hydroxysuccinimide-activated MAG 3 ester for 99m Tc labeling.…”