Polyakov V, Sharma V, Dahlheimer J L, Pica C M, Luker G D, Piwnica-Worms D
Laboratory of Molecular Radiopharmacology, Department of Radiology and Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110, USA.
Bioconjug Chem. 2000 Nov-Dec;11(6):762-71. doi: 10.1021/bc000008y.
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.
将放射性金属配合物快速有效地递送至细胞内部，将使医学成像和放射治疗领域出现新的应用。合成了包含HIV-1 Tat反式激活蛋白序列（GRKKRRQRRR）和合适的基于肽的基序（ε-KGC）的膜渗透性肽缀合物，该基序为螯合锝和铼提供N(3)S供体核心。通过与过金属酸盐、氯化亚锡和葡萄糖庚酸钠的简便转螯合反应制备了锝(V)氧代和铼(V)氧代Tat-肽配合物。反相高效液相色谱显示两种主要的[(99m)Tc]Tat-肽物种（4），其保留时间相差约2分钟，对应于两种[Re]Tat-肽物种（7），显示具有相同的质量，这与两种异构体的形成一致，可能是氧代金属非对映异构体。[(99m)Tc]Tat-肽在体外对转螯合稳定。在人Jurkat细胞中，[(99m)Tc]Tat-肽4以非对映体可比的方式显示出浓缩性细胞积累（比细胞外浓度高30倍）和快速摄取动力学（t(1/2) < 2分钟）。矛盾的是，与37℃相比，在4℃缓冲液中摄取增强，而膜电位去极化以及微管功能和囊泡运输的抑制均无抑制作用。预先加载4的细胞显示出快速洗脱动力学进入无肽溶液。通过缺失N端甘氨酸并进行或不进行生物素化对[(99m)Tc]Tat-肽进行修饰，对净细胞摄取的影响最小。此外，将原型Tat-肽的C端硫醇用荧光素-5-马来酰亚胺标记以产生缀合物8。荧光显微镜直接将缀合物8定位到人Jurkat、KB 3-1和KB 8-5肿瘤细胞的细胞质和细胞核（可能是核仁）中。静脉注射原型[(99m)Tc]Tat-肽4后在小鼠中进行的初步成像研究显示出初始全身分布，并通过肾和肝胆排泄快速清除。对体内小鼠血液和体外人血清的分析显示>95%的复合物完整，而体内小鼠尿液显示65%的母体复合物。因此，这些新型Tat-肽螯合物缀合物能够形成稳定的[Tc/Re(V)]复合物，能快速跨细胞膜转运到细胞内区室，并且可以很容易地进行衍生化，用于分子成像和放射治疗中的进一步靶向应用。
