αB-crystallin elastin-like polypeptides for sustained ocular drug delivery.

The unfolded protein response (UPR) has been mechanistically implicated in multiple ocular disorders, including age-related macular degeneration (AMD). Therapeutic intervention using an αB-crystallin-derived 'mini-cry' chaperone peptide has potential to restore proteostasis; however, its low molecular weight results in rapid ocular clearance. To prolong mini-cry's ocular effects, our team linked it with elastin-like polypeptides (ELPs). ELPs are thermoresponsive polymers that phase-separate into microscale coacervates. Provided ELPs do not interfere with biological activity, cry-ELPs could prolong intraocular retention and improve therapeutic effect. This study compares two cry-ELPs (cry-SI and cry-V96) designed to phase-separate following intravitreal administration. First, chaperone activity was assessed using tau protein aggregation assays, demonstrating that cry-V96 exhibited superior inhibition of tau seeding and fibril disaggregation. Second, modulation of the UPR was assessed in human retinal pigment epithelium cells using tunicamycin-induced stress. Both constructs suppressed PERK/ATF-4/CHOP signaling, with cry-V96 showing the most robust attenuation of ER stress. Third, cellular uptake and subcellular localization were analyzed by biochemical fractionation and Western blotting, revealing enhanced cellular uptake and association and stress-dependent nuclear localization of cry-ELPs, compared to controls. Fourth, the lead construct, cry-V96, was evaluated in vivo in rabbit eyes, where it formed a sustained intravitreal depot, exhibited prolonged retention, and showed no evidence of ocular toxicity by fluorescence imaging and optical coherence tomography (OCT). Finally, proteomic profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 59 proteins selectively enriched by cry-V96, implicating pathways involved in proteostasis, intracellular trafficking, and stress adaptation. Collectively, these findings establish cry-ELPs as multifunctional molecules that combine potent chaperone activity with sustained ocular delivery, providing a promising strategy for targeting UPR dysregulation in AMD and related degenerative diseases.