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Gabriel Baltazar, Sonia Guha, Alan M. Laties, Puneet Tyagi, Uday B. Kompella, Claire H. Mitchell; Acidic Nanoparticles Enhance Degradative Lysosomal Enzyme Activity In Compromised Rpe Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3216.
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Compounds such as N-retinylidene-N-retinyl-ethanolamine (A2E) and chloroquine (CHQ) alkalinize the lysosomes of RPE cells. Alkalinization decreases the activity of pH sensitive degradative enzymes, leading to the incomplete degradation of phagocytic and autophagic material. As we have shown that acidic nanoparticles (NPs) reacidify compromised RPE lysosomes, treatment to reacidify lysosomes may prevent accumulation of autofluorescent material. This study asks if acidic nanoparticles restore the activity of degradative lysosomal enzymes in damaged RPE cells.
Poly DL Lactide nanoparticles were developed. NPs loaded with the fluorescent dye Nile Red were administered to ARPE-19 cells for 30 min and 1 hr to assess the short term colocalization of particles with lysosomes. As cathepsin D is the primary protease in RPE lysosomes, the fluorescent cathepsin D substrate Bodipy FL-pepstatin A was used to determine enzyme activity in situ. ARPE-19 cells were treated with 10 µM CHQ or CHQ + 1 mg/ml NPs for 48 hrs before analyzing cathepsin D activity with a plate reader. To ensure proper localization of Bodipy to the RPE lysosomes, cells were also co-stained with the dye Lysotracker Red and examined microscopically. FACS analysis was used to assess autofluorescence of RPE cells. ARPE-19 cells were exposed to 2 hrs of bovine photoreceptor outer segments (POS), 2 hr chase, followed by a 20 hr application of NPs and/or CHQ. This cycle was repeated daily for 6 days, after which autofluorescence was read using the FITC channel of a FACS machine.
Acidic nanoparticles were rapidly transported to lysosomes; colocalization of red NPs with LysoTracker Green gave a Pearson’s coefficient of 0.31 after 30 min, rising to 0.78 after I hr. Bodipy-FL-pepstatin A also colocalized with LysoTracker Red stain after 30 min of incubation. Cathepsin D activity decreased in cells treated with CHQ, a result consistent with its alkalinizing effect on RPE lysosomes. Application of NPs to CHQ-treated cells restored cathepsin D activity. Finally, treatment with POS and/or CHQ increased RPE cell autofluorescence as seen with FACS analysis. However, treatment with acidic NPs prevented such autofluorescent accumulation.
Acidic nanoparticles can restore degradative function of compromised lysosomes in RPE cells.
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