title: Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.
abstract: 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.
authors.affiliation: Department of Radiology, University of Texas Southwestern Medical Center, 2201 Inwood Road, Dallas, TX, 75390, USA.
authors.forename: Huawei
authors.initials: H
authors.lastname: Cai
authors.forename: Ajay N
authors.initials: AN
authors.lastname: Singh
authors.forename: Xiankai
authors.initials: X
authors.lastname: Sun
authors.forename: Fangyu
authors.initials: F
authors.lastname: Peng
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000473:pathology*; D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D008024:Ligands; D019913:Nuclear Localization Signals; D018719:Erb-b2 Receptor Tyrosine Kinases; C098793:CY5.5 cyanine dye