title: Characterization of novel histidine-tagged Tat-peptide complexes dual-labeled with (99m)Tc-tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy.
abstract: To enable concurrent whole body scintigraphy and direct imaging of subcellular localization of permeation peptides, dual-labeled Tat-peptides useful for both radiometric analysis and fluorescence microscopy are desired for molecular imaging applications. Thus, novel dual-labeled D-Tat-peptides comprising Tat-basic domain (hgrkkrrqrrrgc), C-terminus conjugated with fluorescein-5-maleimide (FM) and N-terminus chelated with [(99m)Tc(CO)(3)] via histidine coordination, were synthesized and characterized. In human Jurkat cells, radiotracer uptake and washout studies revealed concentration-dependent accumulation of the dual-labeled Tat-peptide within cells. Subcellular localization of Tat-peptide was confirmed by fluorescence microscopy using an analogous [Re(CO)(3)] dual-labeled Tat-peptide. As seen with C-terminus single-labeled Tat-peptides, localization to the nucleoli was observed with the dual-labeled Tat-peptide, suggesting that the mechanism of Tat-peptide uptake and localization was not dependent on free peptide termini at either end. In Balb/c mice, biodistribution studies performed with the dual-labeled Tat-peptide showed fluorescence intensity by microscopic analysis that visually confirmed and correlated directly with scintigraphic and radiometric data. Of note, following intravenous administration, little brain penetration of these permeation sequences was observed in vivo. His[(99m)Tc(CO)(3)]-, DTPA[(99m)Tc(CO)(3)]-, and epsilon-lys-gly-cys[(99m)Tc(O)]-labeled Tat-peptides showed significant pharmacokinetic differences in liver and kidney depending on labeling strategy, indicating that Tat-peptide biodistribution can be impacted by the chelation moiety coordinated with (99m)Tc. Thus, we have shown that dual-labeled (99m)Tc-tricarbonyl Tat-peptide-FM conjugates can be conveniently synthesized and enable direct comparison of quantitative radiometric and qualitative fluorescence data both in vitro as well as in vivo.
authors.affiliation: Molecular Imaging Center, Mallinckrodt Institute of Radiology, Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110, USA.
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Bullok
authors.forename: Mary
authors.initials: M
authors.lastname: Dyszlewski
authors.forename: Julie L
authors.initials: JL
authors.lastname: Prior
authors.forename: Christina M
authors.initials: CM
authors.lastname: Pica
authors.forename: Vijay
authors.initials: V
authors.lastname: Sharma
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D000818:Animals; D002614:Chelating Agents; D019793:Fluorescein / Q000032:analysis*; D015696:Gene Products, tat / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics; D006639:Histidine / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019169:Jurkat Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D015609:Organotechnetium Compounds / Q000032:analysis*; D004369:Pentetic Acid; D010446:Peptide Fragments / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics; D021381:Protein Transport; D011877:Radionuclide Imaging / Q000379:methods*; D019275:Radiopharmaceuticals / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: D002614:Chelating Agents; D015696:Gene Products, tat; D015609:Organotechnetium Compounds; D010446:Peptide Fragments; D019275:Radiopharmaceuticals; D006639:Histidine; D004369:Pentetic Acid; D019793:Fluorescein