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RA Bejjani, D BenEzra, J-LL Bourges, S Gautier, M Halhal, D Chauvaud, R Gurny, FF Behar-Cohen; Phagocytosis of Polylactides (PLA) Nanoparticles by Bovine and Human RPE Cells In Vitro . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2295.
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Purpose: To study the ability of bovine (BRPE) and human retinal pigment epithelial cells (HRPE) to phagocyte in-vitro PLA NPs and the effect of these ingested particles on cell viability and proliferation. Methods: Human and bovine RPE cells were sub cultured and incubated with various concentrations (0.01, 0.1, 1, 4, 8 mg/ml) of PLA NPs loaded with Rhodamine (Rh) 6G. NPs were added either at cell confluence or during the proliferating phase. Added NPs were washed away from the RPE cultures after 24 hours or left in contact with the cells in culture for a varying length of time. Control cultures for comparison were incubated similarly with the same concentrations of free Rh or blank PLA NPs. The extent of NPs phagocytosis by the cultured RPE cells was assessed by histology, phase and fluorescent microscopy. Cell viability was assessed using trypan blue and the MTT test . Cell morphology was examined using inverted microscope and phase contrast. DAPI staining was used for evaluation of cell nuclei at 1, 4 and 8 days. Cell proliferation was measured by cell counting at the same time points. Results: No significant differences between BRPE and HRPE cells were detected in any of the evaluated parameters. Few NPs were observed within the RPE cells cytoplasm already 6 hours after incubation. The number of ingested particles per cell and the relative number of ingesting RPE cells within the cultures increased in parallel with higher NP concentrations reaching a plateau at 1 mg/ml. Concentrations of NPs as high as 8 mg/ml did not induce any significant RPE cell toxicity nor they interfered with their proliferative abilities. At all tested time points, the number of cells per culture and the MTT values during the first 5 days in culture were similar when cultures with loaded NPs were compared to the control cultures incubated with blank NPs, free rhodamine or saline. Conclusion: RPE cells have a high potential for NPs internalization. Ingested NPs are not toxic for cultured BRPE or HRPE. Targeting functional gene fragment delivery to RPE cells using this methodology may be an appealing approach for potential gene therapy.
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