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Tools that restore lysosomal pH to an optimal acidic range could enhance enzymatic degradation and reduce waste accumulation. Acidic nanoparticles offer a promising strategy for restoring lysosomal function, but accurate tracking of organelle delivery and long-term retention is needed to optimize dosage. To improve detection and enhance delivery, nanoparticles were synthesized from Poly(D,L-lactide-co-glycolide) (PLGA) polymers covalently linked to the fluorescent Cyanine3 amine (Cy3) probe. Nanoparticle concentration and loading times were optimized to achieve &gt;90% delivery to lysosomes in cultured induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells. Uptake was heterogeneous, varying between adjacent cells. Once loaded into lysosomes, the nanoparticles were stably retained, with no detectable changes in concentration, distribution, or size for at least 28 days. iPS-RPE cells internalized more nanoparticles than the ARPE-19 cell line or mouse optic nerve head astrocyte cultures. Functionally, PLGA nanoparticles restored an acidic pH and cathepsin D levels in compromised lysosomes. In summary, Cy3-PLGA nanoparticles enabled improved tracking and long-term delivery to lysosomes, supporting future\n                  <jats:italic>in vivo<\/jats:italic>\n                  applications to restore lysosomal pH in aging and degenerating tissues.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:p>\n                    Increased lysosomal pH reduces degradative enzyme efficiency and contribute to age-dependent neurodegeneration. This study describes synthesis of nanoparticles to restore an acidic lumen and degradative function. Nanoparticles were optimized for lysosomal delivery to astrocytes and iPS-derived retinal pigmented epithelial (RPE) cells. The fluorescent marker Cy3 was covalently bound to polymers for improved tracking to lysosomes. Particles were stably retained inside the lysosomal lumen for at least 28 days. Nanoparticles restored pH to compromised lysosomes to baseline levels and increased active Cathepsin D. The improved design will aid\n                    <jats:italic>in vivo<\/jats:italic>\n                    tracking and repair in models where lysosomal alkalinization contributes to pathology. Created in BioRender. Mitchell, C. (2025)\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/BioRender.com\/8hvj96m\">https:\/\/BioRender.com\/8hvj96m<\/jats:ext-link>\n                    .\n                  <\/jats:p>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"659991v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>New and Noteworthy<\/jats:title>\n                  <jats:p>Tools that restore acidic pH in compromised lysosomes can enhance autophagy and waste degradation in degenerative disorders marked by excessive accumulation. Here, we describe the novel synthesis of lysosome-targeted nanoparticles composed of PLGA polymers covalently bound to Cy3 fluorescent dye. These Cy3-PLGA nanoparticles enabled improved tracking of lysosomal delivery and demonstrated sustained long-term retention within lysosomes, supporting their potential for future applications to restore lysosomal pH in aging and degenerating tissues.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.06.16.659991","type":"posted-content","created":{"date-parts":[[2025,6,17]],"date-time":"2025-06-17T07:15:16Z","timestamp":1750144516000},"source":"Crossref","is-referenced-by-count":0,"title":["Sustained Lysosomal Delivery of Enhanced Cy3-Labeled Acid Nanoparticles Restores Lysosomal pH in Retinal Pigment Epithelial Cells and Astrocytes"],"prefix":"10.64898","author":[{"given":"Jiaqi","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianchen","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wennan","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davit","family":"Jishkariani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Tsourkas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Kaja","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rohini M.","family":"Nair","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua L.","family":"Dunaief","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire H.","family":"Mitchell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025061804450843000_2025.06.16.659991v1.1","doi-asserted-by":"publisher","DOI":"10.1007\/s13311-019-00742-3"},{"key":"2025061804450843000_2025.06.16.659991v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbapap.2020.140443"},{"key":"2025061804450843000_2025.06.16.659991v1.3","unstructured":"Barrett A. 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