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, Gαq, and β-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, Gαq, and β-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.