Cell-compatible, integrin-targeted cryptophane-129XeNMR biosensors

Abstract

Peptide-modified cryptophane enables sensitive detection of protein analytes using hyperpolarized 129Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing αvβ3 integrin 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 αvβ3 integrin 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 hyperpolarized 129Xe 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 αvβ3 integrin and fibrinogen binding to αIIbβ3 integrin. Functionalization of the conjugate with two propionic acid groups improved water solubility for hyperpolarized 129Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the 129Xe-cryptophane–cyclic RGDyK biosensor. Introduction of αIIbβ3 integrin receptor in detergent solution generated a new “bound” 129Xe biosensor peak that was shifted 4 ppm downfield from the “free” 129Xe biosensor.

Measurement of radon and xenon binding to a cryptophane molecular host

“…Thus, the design of higher affinity xenon-binding molecules such as TTEC extends the range of possible biological applications. Biologically targeted cryptophanes are under development as potential 129 Xe MRI contrast agents, as demonstrated by xenon biosensors for the prototypical biotin-avidin interaction (53,54) and other proteins (55)(56)(57), as well as preliminary cell studies (58)(59)(60). This demonstration of radon binding to cryptophane raises similar possibilities of molecularly functionalized radon binders for biological, environmental, and materials applications involving radon delivery, sequestration, or detection.…”

NMR-Active Nuclei for Biological and Biomedical Applications

“…Cryptophane was functionalised with cyclic RGDyK peptide and 129 Xe NMR revealed a single resonance at 67 ppm for the 129 Xe-cryptophanecyclic RGDyK biosensor. Introduction of αIIb 3 integrin receptor in detergent solution generated a new 'bound' 129 Xe biosensor signal that was shifted 4 ppm downfield from the 'free' 129 Xe biosensor [323]. A 'clickable' and highly water-soluble PEGylated cryptophane has been developed as a potential universal platform for 129 Xe NMR biosensors.…”

NMR Hyperpolarization Techniques of Gases

“…[153] As part of these studies, cell uptake and toxicity evaluations set the bar for target concentrations of functionalized hosts. [148,154] Xe itself passes the cell membrane [149] and does not require further attention to reach intracellular targets. Cell-penetrating peptides proved to be a valuable measure for achieving micromolar intracellular concentrations.…”

“…To date, cryptophane-based sensors that use specific binding to the target have been implemented for sensing the enzyme MMP7, [142] nucleotides, [143] human carbonic anhydrase, [144] MHC class II, [145] zinc ions, [146] the glycoprotein CD14 [147] and the receptors for integrin, [148] transferrin, [149] EGF, [150] and folate. [151] An indirect binding approach was pursued through in situ click chemistry with metabolically labeled cell surface glycans.…”