Characterization of Novel Histidine-Tagged Tat-Peptide Complexes Dual-Labeled with ^99mTc-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.

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).…”

TAT-BH4 and TAT-Bcl-xL Peptides Protect against Sepsis-Induced Lymphocyte Apoptosis In Vivo

“…Thus, there is no endotoxin contamination in TAT-BH4 peptide. It is important to note that both of these compounds should be delivered to most cells in the body, but penetration across the blood brain barrier may be limited (33,35,36). Although lymphocytes are a primary target of sepsis-induced apoptosis, the gastrointestinal epithelial cells also undergo a large acceleration in apoptosis during sepsis (3,14).…”

“…To prepare the fluorescent labeled TAT-BH4, (d) Ac-C (FM) RKKRR-Orn-RRR-␤-A-(l)-SNRELVVDFLSYKLSQKGYS-COOH, an N terminus cysteine, was included in the initial solid state peptide synthesis of the peptide and, following HPLC purification, the peptide was thiol conjugated to fluorescein maleimide (1.2 equivalent; Molecular Probes) at ambient temperature in 50% dimethylformamide/water for 2 h. Quantitative yields were analyzed by C 18 reverse-phase HPLC. For labeling, cells were suspended for 30 min in modified Earl's balanced salt solution containing 1 M fluorescently labeled TAT-BH4 (33). Control cells were treated identically, except no labeled TAT-BH4 was added.…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…In a previous study, however, no PTD mediated transduction in brain was observed, [46] possibly because beta-gal was chemically conjugated to PTD instead of recombinant fusion protein. Similarly, another study also failed to deliver PTD- 99 Tc to the brain [76]. Since PTD is conjugated to Tc or beta-gal, this might have resulted in structural modifications in the PTD and hence no transduction was observed.…”