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 (PAR2); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to selectively antagonize intracellular PAR2 signaling of pain. PAR2 agonists evoked sustained activation of PAR2, Gq and {beta}-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 PAR2, Gq and {beta}-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 PAR2 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 PAR2 signals in colonocytes and nociceptors mediate pain. Antagonism of PAR2 in organelles, rather than at the plasma membrane, provides effective pain relief. Significance StatementOnce activated at the cell surface, many GPCRs internalize and continue to signal. The mechanisms and physiological relevance of intracellular GPCR signaling are uncertain. By using organelle-targeted biosensors, we detected sustained activation of the GPCR, PAR2, and its effectors in early, late and recycling endosomes, the cis- and trans-Golgi apparatus, and the cytosol and nucleus. NPs that delivered AZ3451, a PAR2 antagonist, to endosomes disrupted these intracellular signals, whereas unencapsulated AZ3451 was minimally effective. After intracolonic administration to mice, NPs accumulated in colonocytes and neurons. NP-encapsulated AZ3451, but not unencapsulated AZ3451, reversed pain in preclinical models of inflammatory bowel disease. Thus, intracellular PAR2 signaling mediates pain and antagonism of intracellular rather than plasma membrane PAR2 provides effective therapy.