Journal of Fluorescence

, Volume 25, Issue 1, pp 113–117 | Cite as

Synthesis and Characterization of Her2-NLP Peptide Conjugates Targeting Circulating Breast Cancer Cells: Cellular Uptake and Localization by Fluorescent Microscopic Imaging

  • Huawei Cai
  • Ajay N. Singh
  • Xiankai Sun
  • Fangyu Peng


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.


Breast cancer Her2/neu oncoprotein Circulating tumor cells Nuclear localization sequence peptide Radionuclide cancer therapy 



This research project was supported by the Department of Defense (CDMRP/BCRP, W81XWH-11-1-0188 to FP) and Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center).

Conflict of Interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Huawei Cai
    • 1
    • 4
  • Ajay N. Singh
    • 1
    • 5
  • Xiankai Sun
    • 1
    • 2
    • 3
  • Fangyu Peng
    • 1
    • 2
    • 3
  1. 1.Department of RadiologyUniversity of Texas Southwestern Medical CenterDallasUSA
  2. 2.Advanced Imaging Research CenterUniversity of Texas Southwestern Medical CenterDallasUSA
  3. 3.Harold C. Simmons Comprehensive Cancer CenterUniversity of Texas Southwestern Medical CenterDallasUSA
  4. 4.Department of Nuclear Medicine, West China HospitalSichuan UniversityChengduPeople’s Republic of China
  5. 5.Department of Pharmaceutical ScienceAppalachian College of PharmacyOakwoodUSA

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