title: Cell-compatible, integrin-targeted cryptophane-129Xe NMR biosensors.
abstract: Peptide-modified cryptophane enables sensitive detection of protein analytes using hyperpolarized129Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing αvβ3integrin receptor, which is overexpressed in many human cancers. Cryptophane was functionalized with cyclic RGDyK peptide and Alexa Fluor 488 dye, and cellular internalization was monitored by confocal laser scanning microscopy. Competitive blocking assays confirmed cryptophane endocytosis through an αvβ3integrin receptor-mediated pathway. The peptide-cryptophane conjugate was determined to be nontoxic in normal human lung fibroblasts by MTT assay at the micromolar cryptophane concentrations typically used for hyperpolarized129Xe NMR biosensing experiments. Flow cytometry revealed 4-fold higher cellular internalization in cancer cells overexpressing the integrin receptor compared to normal cells. Nanomolar inhibitory concentrations (IC50= 20-30 nM) were measured for cryptophane biosensors against vitronectin binding to αvβ3integrin and fibrinogen binding to αIIbβ3integrin. Functionalization of the conjugate with two propionic acid groups improved water solubility for hyperpolarized129Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the129Xe-cryptophane-cyclic RGDyK biosensor. Introduction of αIIbβ3integrin receptor in detergent solution generated a new "bound"129Xe biosensor peak that was shifted 4 ppm downfield from the "free"129Xe biosensor.
authors.affiliation: University of Pennsylvania, Department of Chemistry, 231 South 34 St, Philadelphia, PA 19104-6323, USA.
authors.forename: Garry K
authors.initials: GK
authors.lastname: Seward
authors.affiliation: University of Pennsylvania, Department of Chemistry, 231 South 34 St, Philadelphia, PA 19104-6323, USA.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Bai
authors.affiliation: University of Pennsylvania, Department of Chemistry, 231 South 34 St, Philadelphia, PA 19104-6323, USA.
authors.forename: Najat S
authors.initials: NS
authors.lastname: Khan
authors.affiliation: University of Pennsylvania, Department of Chemistry, 231 South 34 St, Philadelphia, PA 19104-6323, USA.
authors.forename: Ivan J
authors.initials: IJ
authors.lastname: Dmochowski