Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis. Methods: Acid-sensitive cyclic peptide c[E₄W₅C] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with ⁶⁴Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [⁶⁴Cu]Cu-c[E₄W₅C] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non-pH-sensitive cell-penetrating control peptide (c[R₄W₅C]) was used to confirm specificity of [⁶⁴Cu]Cu-c[E₄W₅C]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [⁶⁴Cu]Cu-c[E₄W₅C] PET signal. Results: [⁶⁴Cu]Cu-c[E₄W₅C] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/nmol. Significantly increased [⁶⁴Cu]Cu-c[E₄W₅C] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines (P < 0.001-0.05). Additionally, there was significantly increased [⁶⁴Cu]Cu-c[E₄W₅C] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [⁶⁴Cu]Cu-c[R₄W₅C] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [⁶⁴Cu]Cu-c[E₄W₅C] tracer. Conclusion: Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [⁶⁴Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

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