Nanomedicines targeting signaling of protease-activated receptor 2 in organelles provide sustained analgesia

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of GPCR signaling in organelles 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 subcellular compartments were used to analyze organelle-specific signaling of protease-activated receptor 2 (PAR ₂ ); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to selectively antagonize intracellular PAR ₂ signaling of pain. PAR ₂ agonists evoked sustained activation of PAR2, Gαq and β-arrestin-1 in early, late and recycling endosomes and the cis - and trans . Golgi apparatus, and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with organelle-targeted biosensors. Dendrimer and core-shell polymeric NPs accumulated in early and late endosomes of HEK293 cells, colonic epithelial cells and nociceptors, detected by confocal imaging of fluorescent NPs. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR2 antagonist. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR ₂ , Gαq and β-arrestin-1 activation in endosomes and the Golgi apparatus and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs targeted neurons, sites of PAR ₂ expression. Both NP-AZ3451 formulations, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. Thus, organelle-specific PAR ₂ signals in colonocytes and nociceptors mediate pain. Antagonism of PAR ₂ in organelles, rather than at the plasma membrane, provides effective pain relief.