title: Ultrasound molecular imaging of E-selectin in tumor vessels using poly n-butyl cyanoacrylate microbubbles covalently coupled to a short targeting peptide.
abstract: OBJECTIVE
  The purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin-specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR.
  
  
  METHODS
  The E-selectin-specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate-stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-α-stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression.
  
  
  RESULTS
  Success of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin-specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05).
  
  
  CONCLUSIONS
  Clinically translatable microbubbles that were covalently coupled to the short E-selectin-specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.
authors.affiliation: From the *Department of Experimental Molecular Imaging, University Clinic and Helmholtz Center for Biomedical Engineering, RWTH Aachen University, Aachen, Germany; †Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; ‡Institute for Molecular Cardiovascular Research, University Clinic, RWTH Aachen University, Aachen; and §Academic Radiology Baden-Baden, Diagnostic and Interventional Radiology, University Medical Center Heidelberg, Baden-Baden, Germany.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fokong
authors.forename: Ana
authors.initials: A
authors.lastname: Fragoso
authors.forename: Anne
authors.initials: A
authors.lastname: Rix
authors.forename: Adelina
authors.initials: A
authors.lastname: Curaj
authors.forename: Zhuojun
authors.initials: Z
authors.lastname: Wu
authors.forename: Wiltrud
authors.initials: W
authors.lastname: Lederle
authors.forename: Olga
authors.initials: O
authors.lastname: Iranzo
authors.forename: Jessica
authors.initials: J
authors.lastname: Gätjens
authors.forename: Fabian
authors.initials: F
authors.lastname: Kiessling
authors.forename: Moritz
authors.initials: M
authors.lastname: Palmowski
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry; D019040:E-Selectin / Q000378:metabolism*; D004659:Enbucrilate / Q000737:chemistry*; D005260:Female; D051379:Mice; D008819:Mice, Nude; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods*; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism*; D010455:Peptides / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014463:Ultrasonography / Q000379:methods*
chemical_list: D004337:Drug Carriers; D019040:E-Selectin; D010455:Peptides; D004659:Enbucrilate