Elevated extracellular acidity due to increased glycolytic flux is a hallmark of many pathological states. Exploiting acidosis to develop probes that monitor and track inflammation is a viable approach with the flexibility to range across a spectrum of toxicologically-relevant endpoints, from bioenergetic measurements to tumor diagnosis. pH-Low-Insertion-Peptide (pHLIP) is a pH-sensitive peptide that is capable of cellular transmembrane insertion through a helix formation mechanism that is reliant on the protonation of aspartic acid residues under localized acidic conditions. Upon insertion, the C-terminus translocates inside the cell while the N-terminus remains extracellular. By tethering a fluorophore to the N-terminus, researchers may visualize inflammatory response in real time, which provides an orthogonal view to complement the findings of cytokine profiling <i>in vivo. In vitro </i>studies using pHLIP-1 allow for the targeting and sorting of metabolically compromised cells for further study. Our laboratory has been optimizing pHLIP variants coupled to FITC to demonstrate successful targeting of L6 rat skeletal myoblasts post-exposure to oligomycin. Our techniques have achieved 100% localization to acidic cells in under one minute. Fluorescence polarization aggregation studies have been performed with our variant to pinpoint the optimal conditions for localization and insertion in a short period of time. Understanding the precise kinetic mechanism is the key to many toxicological applications, so a range of variants continue to be explored to fine tune insertion properties.

pHLIP-dye conjugates as probes for visualizing inflammatory response

254th National Meeting (2017)

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