Nanomedicines Targeting Protease-activated Receptor 2 in Endosomes Provide Sustained Analgesia

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of intracellular GPCR signaling are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically-encoded biosensors targeted to the plasma membrane and early endosomes were used to analyze compartmentalized signaling of protease-activated receptor 2 (PAR₂); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to preferentially antagonize intracellular PAR₂ signaling of pain. PAR₂ agonists evoked sustained activation of PAR₂, Gaq and b-arrestin-1 in early endosomes and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with targeted biosensors. Fluorescent dendrimer and core-shell polymeric NPs accumulated in endosomes of HEK293T cells, colonic epithelial cells and nociceptors, detected by confocal microscopy. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR₂ modulator. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR₂, Gaq and b-arrestin-1 activation in early endosomes and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, fluorescent dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs accumulated in neurons, sites of PAR₂ expression. Both NP formulations of AZ3451, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. These results provide evidence that PAR₂ endosomal signaling mediates pain and that nanomedicines that antagonize PAR₂ in endosomes effectively relieve pain. NP-mediated delivery may improve the efficacy of other GPCR antagonists for treatment of diverse diseases. There is a pressing need to develop non-opioid treatments for pain. In the diseased colon, proteases that activate PAR₂ on nociceptors and colonocytes evoke pain. Since PAR₂ endosomal signaling underlies pain, PAR₂ in endosomes is a relevant therapeutic target. We leveraged the predisposition of NPs to accumulate in endosomes to deliver the PAR₂ antagonist, AZ3451, to intracellular sites of pain signaling. NPs that delivered AZ3451 to endosomes effectively reversed PAR₂ endosomal signals, whereas unencapsulated AZ3451 was minimally effective. When administered into the colonic lumen of mice, NP-encapsulated AZ3451, but not unencapsulated AZ3451, reversed pain and normalized aberrant behavior in preclinical models of inflammatory bowel disease. Nanomedicines that block intracellular signaling of PAR₂, and possibly other GPCRs, effectively relieve visceral pain.