PLGA nanoparticles restore acidic pH and degradative function to compromised lysosomes with Cy3-labeling providing enhanced tracking to lysosomes

Lysosomal dysfunction and elevated lysosomal pH are hallmark features of age-related neurodegenerative diseases including age-related macular degeneration (AMD), Alzheimer's disease (AD), and Parkinson's disease (PD). Restoring lysosomal acidity is important for maintaining enzymatic degradation, preventing protein aggregation, and reducing cellular waste accumulation in degenerating tissues. Acidic nanoparticles represent a promising therapeutic strategy to normalize lysosomal pH; however, accurate monitoring of their delivery, retention, and dosage is critical for rigorous evaluation. To address this, we developed fluorescently labeled poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles conjugated with Cyanine3 amine (Cy3). Nanoparticle uptake was systematically optimized, achieving over 90% delivery to lysosomes of induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells, although uptake rates varied among adjacent cells. Once internalized, nanoparticles demonstrated remarkable stability, with no detectable change in concentration, distribution, or size for at least 28 days. iPS-RPE cells exhibited higher nanoparticle internalization compared with the ARPE-19 cell line and optic nerve head astrocytes. The capacity of the nanoparticles to restore function to stressed lysosomes was confirmed by their ability to reacidify lysosomes, restore cathepsin B activity, and increase the levels of active cathepsin D. The nanoparticles also reduced the levels of LC3II in astrocytes treated with chloroquine, indicating that they can also restore autophagy rates. In summary, this study demonstrates the value of Cy3 labeling for enhanced nanoparticle tracking to lysosomes. The findings also identify PLGA nanoparticles as powerful tools for restoring degradative lysosomal function and autophagy in cells undergoing lysosomal stress.NEW & NOTEWORTHY Tools that restore acidic pH in compromised lysosomes can enhance autophagy and waste clearance in degenerative disorders characterized by excessive accumulation. Here, we describe the synthesis of lysosome-targeted nanoparticles composed of poly(d,l-lactide-co-glycolide) (PLGA) polymers covalently bound to the fluorescent dye Cyanine3 amine (Cy3). These Cy3-PLGA nanoparticles enable precise tracking of lysosomal delivery and demonstrate sustained long-term retention within lysosomes, supporting their potential for future applications aimed at restoring lysosomal pH in aging and degenerating diseases.