Nanomedicines Targeting Endosomal PAR2 Signaling Reverse Colonic Inflammatory Pain

Background & Aims

Abdominal inflammatory pain is a common and persistent symptom of inflammatory bowel disease (IBD). Pain can arise from different mechanisms and persists due to changes in afferent sensory neurons activating threshold. A key role in this process is played by a protease-activated receptor 2 (PAR2), a G-Protein-Coupled Receptor, which is activated by proteases in the inflamed colon and internalizes generating sustained signals from endosomes1. PAR2 is expressed in colonocytes, inflammatory cells, and sensory neurons and when internalized participates in the progression of inflammation and generation of neuronal hyperexcitability. Nanomedicines offer the possibility to specifically target disease tissue, cells, and organelles2 and inhibit receptors to signal from endosomes which may lead to sustained nociceptive signals.
Slow-release dendrimer and polymeric nanoparticles encapsulating a PAR2 antagonist accumulate in endosomes blocking PAR2 signaling and reducing inflammatory pain.

Methods

Nanoparticle characterization, uptake, and trafficking were assessed in HEK293T, T84, nociceptors (sensory neurons in the dorsal root ganglia), and on colonic mouse tissue (ex-vivo and in-vivo) using bioluminescence energy transfer assays, patch clamp, and microscopy respectively. IBD-like models of Inflammatory pain (TNBS and DSS colitis) were induced in 10-12 weeks old male C57BL/6J mice. Pain and inflammation were measured using the mechanical allodynia von Frey test and qRT-PCR respectively. Non-evoked pain behavior was assessed using automated behavioral instruments (mouse spectrometer). Nanoparticles encapsulating PAR2 antagonists, AZ3451, were used to specifically target the receptor in endosomes. Experimental groups received, AZ3451, AZ3451 encapsulated into nanoparticles, empty nanoparticles, and vehicles.

Results

In HEK293T, T84 cells, and dorsal root ganglia culture nanoparticles (PAMAM-Chol-cy5 and PEG-Poly-QD) accumulated in early endosomes within 30 min and were retained for up to 4 h.
In an ex-vivo colonic tissue and in vivo enema injection nanoparticle uptake was preferentially in mucosal colonocytes for the dendrimer nanoparticles and mucosal colonocytes and enteric nervous tissue for the polymeric nanoparticles. Nanoparticles were retained in the colonic tissue for more than 8 h.
Nanoparticles encapsulating AZ3451 inhibit the assembly of the signalosome (PAR2/G?/?-arrestin) in the endosome and Golgi apparatus inhibiting neuronal hyperexcitability, while unconjugated AZ3451 or empty nanoparticles failed to inhibit signalosome assembly and neuronal hyperexcitability.
In animal models of colonic lumen delivery of nanoparticles encapsulating AZ3451 greatly reduces allodynia, pro-inflammatory cytokine levels, and normalized locomotor, exploratory, and grooming behaviors.

Conclusions

PAR2 internalizes in the inflamed colon, likely due to the activation of protease, and endosomal signaling in colonocytes and nociceptors mediates the proinflammatory and pronociceptive actions of PAR2, suggesting that PAR2 in endosomes is a target for therapy for a condition like inflammatory bowel disease1,3. One-third of the commercially available drugs, target GPCRS. However, those drugs target the receptor on the plasma membrane, which we showed to be highly inefficient and poorly efficacious in inhibiting sustained pain and inflammation. Therefore, luminally administered nanoparticles provide an opportunity to target the subcellular-specific signals in colonocytes and neurons that mediate disease, minimizing dosing and the side effects of systemic exposure.

References

1 Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome
Nestor N. Jimenez-Vargas et al. PNAS. 2018.
2 Nanomedicine in cancer therapy. Dahua Fan et al. Nature2022.
3 Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain. Latorre R et al. PNAS 2022