Cai Huawei, Singh Ajay N, Sun Xiankai, Peng Fangyu
Department of Radiology, University of Texas Southwestern Medical Center, 2201 Inwood Road, Dallas, TX, 75390, USA.
J Fluoresc. 2015 Jan;25(1):113-7. doi: 10.1007/s10895-014-1486-9. Epub 2015 Jan 27.
To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.
合成一种由Her2/neu靶向肽和核定位序列肽（NLP）组成的荧光Her2-NLP肽偶联物，并评估其对放射性核素癌症治疗中靶向Her2/neu阳性循环乳腺癌细胞（CBCC）的细胞摄取和细胞内定位。通过将由Her2结合肽（NH2-GSGKCCYSL）和NLP肽（CGYGPKKKRKVGG）组成的二价肽序列通过具有6个重复单元的聚乙二醇（PEG）链连接，并与活化的Cy5.5酯偶联，合成荧光Cy5.5 Her2-NLP肽偶联物。通过高效液相色谱法分离和纯化该偶联物，然后通过基质辅助激光解吸电离质谱（Maldi-MS）进行表征。在分别将Cy5.5-Her2-NLP与Her2/neu阳性乳腺癌细胞和Her2/neu阴性对照乳腺癌细胞孵育后，使用共聚焦显微镜通过荧光显微镜成像评估荧光Cy5.5 Her2-NLP肽偶联物的细胞内定位。在体外将乳腺癌细胞与Cy5.5-Her2-NLP偶联物孵育后，在Her2/neu阳性乳腺癌细胞（SKBR-3和BT474细胞系）的细胞质中检测到荧光信号，但在Her2/neu阴性乳腺癌细胞（MDA-MB-231）的细胞质中未检测到或检测到很少的荧光信号。在用Cy5.5-Her2-NLP肽偶联物孵育的Her2/neu阳性SKBR-3和BT474乳腺癌细胞核内以及Her2/neu阴性MDA-MB-231细胞内均未检测到荧光信号，这表明Cy5.5-Her2-NLP偶联物在被Her2/neu阳性乳腺癌细胞摄取和内化后定位于细胞质中，其核定位较差。Her2结合肽（KCCYSL）是使用β(-)或发射α粒子的放射性核素对Her2/neu阳性乳腺癌进行放射性核素治疗的一种有前景的药物，但Her2-NLP肽偶联物较差的核定位可能会限制其使用I-125或其他俄歇电子发射放射性核素根除Her2/neu阳性CBCC的应用。
