Acidic nanoparticles are trafficked to lysosomes and restore an acidic lysosomal pH and degradative function to compromised ARPE-19 cells

Abstract

Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.

Figure 1. Acid nanoparticles are delivered to lysosomes of ARPE-19 cells.

A-D. Live cell images of colocalized nanoparticles with ARPE-19 lysosomes after a 24 hour incubation with NP2R A. ARPE-19 cell lysosomes as visualized with the dye LysoTracker Green. Images taken at 40x magnification with a Zeiss confocal microscope. LysoTracker detected at 488/500 nm (excitation/emission). B. ARPE-19 ingestion of Nile Red stained nanoparticle NP2R. Cells were incubated for 24 hours in full culture medium with 1 mg/ml concentration of NPs. Nanoparticles were detected at 540/580 (excitation/emission). Before incubation, the nanoparticle suspension was passed through a 0.8 µM syringe filter to remove clumped particles. After incubation period, cells were washed thoroughly with isotonic solution in an attempt to further remove clumps and any extracellular NPs. C. DIC image of the ARPE-19 cells and nanoparticles. With this image the morphology of the ARPE-19 cells are clearly visible. D. Composite image of the LysoTracker Green (lysosomes), Nile Red (nanoparticles), and DIC exposures. This image demonstrates the colocalization of the ingested nanoparticles with the ARPE-19 lysosomes. E. Concentration dependence of lysosomal delivery. The degree of colocalization of NP1R, NP2R, and NP3R with lysotracker green as a function of concentration. Each point is the mean +/− SEM of a calculated Pearson’s coefficient within one area of interest (AOI) in one microscope field; n = 3 fields. Inserts indicate overlap of NP1R and NP3R. In 1E, NP2R were incubated for 1 hr. before colocalization was determined.

Figure 2. Rapid delivery of nanoparticles to lysosomes.

A. Composite image of ARPE-19 cells with internalized NP3R nanoparticles (red, 1 mg/ml) and Lysotracker (green) after 15 min incubation. B. NP3R and lysosomes after 30 min incubation. C. NP3R and lysosomes after 1 hr. incubation. Bar  = 10 µM in panels A-C. D. Colocalization of NP3R nanoparticles with ARPE-19 cells as a function of time. Each point is the mean +/− standard deviation of the Pearson’s coefficient in one microscope field; n = 3. Error bars are present but too small to be detected for the 15 min point.

Figure 3. Nanoparticles lower lysosomal pH.

A. While NP1 did not alter baseline levels of lysosomal pH (pHL), NP2 and NP3 acidified the lysosomes significantly. Lysosomal pH was measured 1 hr. after addition of nanoparticles. Here and throughout the figure, nanoparticles were given at 1 mg/ml. n = 8. * p<0.05 vs. control, ANOVA on ranks, Dunn’s posthoc test. B. Chloroquine (CHQ; 10 µM) raised the lysosomal pH. NP2 and NP3 significantly lowered lysosomal pH, while NP1 had little effect. Chloroquine and nanoparticles were applied concurrently 1 hr. before pH measurements. *p<0.001 vs. control, **p<0.001 vs. CHQ, ANOVA with Tukey post hoc test. n = 8. C. NP2 and NP3 induced sustained acidification of lysosomal pH in cells treated with 10 µM chloroquine. The acidification decreased with time but was still detected. Chloroquine and nanoparticles were added on day 1 and remained in the bath without a solution change. The effect of the nanoparticles was defined as their relative effectiveness at bringing lysosomal pH towards baseline; the absolute numbers did vary somewhat but this normalization accounted for such differences. % Reacidification  = 100*((CHQ-(CHQ+NP))/(CHQ-Control)). Data from 31 plates.

Figure 4. BODIPY FL-pepstatin A: Probing cathepsin D activity.

A. Lysosomes of ARPE-19 cells as stained with LysoTracker Red B. BODIPY FL-pepstatin A staining. C. Composite image demonstrating colocalization of BODIPY fluorophore with RPE lysosomes D. CHQ administration raises the lysosomal pH, inactivating cathepsin D and hindering the binding of the BODIPY probe to the enzyme. This is seen quantitatively as a lowering in the amount of fluorescence (measured in arbitrary light units, ALU). Nanoparticles reverse this process, with NP3 significantly restoring cathepsin D activity. * p<0.05 vs. control, ** p<0.05 vs. CHQ; one-way ANOVA with Tukey post hoc test. n = 5 wells from 1 plate. Similar results seen in 2 plates.

Figure 5. Nanoparticles reduce autofluorescence and opsin levels associated with ingestion of photoreceptor outer segments.

A. Sample readout of the FACS analysis demonstrating treatment with NP3 greatly reduced the mean autofluorescence at 488 nm in RPE cells treated with chloroquine (CHQ) and phagocytosed photoreceptor outer segments (POS). B. Nanoparticles reduced the autofluorescence in ARPE-19 cells given POS, CHQ, and or POS+CHQ. Bars represent the mean ± SEM of autofluorescence detected at 488 nm. * p<0.05 vs. control; # p<0.05 vs. POS+CHQ. ANOVA. C. Immunoblot for opsin detected in ARPE-19 cells in the absence of photoreceptor outer segments (Control), after exposure to outer segments over 7 days (POS), and with a delayed addition of PLA NP3 after each outer segment feeding (POS+NP3). The blot was at the predicted size of ∼40 kDa. GAPDH binding of the blot is demonstrated below. D. Quantitation of opsin levels in immunoblots. Levels were first controlled for GAPDH staining, and then normalized to the mean POS value in each blot to control for variation. * p<0.001, n = 4.

Figure 6. Model of enhanced photoreceptor degradation.

A. RPE cells with compromised lysosomes cannot sufficiently degrade photoreceptor outer segments. The undigested material accumulates inside the cell as autofluorescent lipofuscin. B. After treatment with acidic nanoparticles, RPE lysosomes are more capable of breaking down the POS. The end result is a substantial decrease in undigested debris and lipofuscin.