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M. E. Boulton, A. Paul, M. Davies, H. Vittal Rao, M. Rozanowska, R. Duncan; A Nanomedicine Strategy for the Removal of Lipofuscin From the RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4189.
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The aim of this study was to determine if a bioresponsive nanomedicine could be tailored for the removal of lipofuscin from RPE cells. To test this we conjugated the phase II detoxification enzyme, glutathione S-transferase (GST) to a succinylated dextrin polymer.
Purity of the conjugate was confirmed by FPLC and gel electrophoresis and activity determined using a fluorimetric assay kit. For cellular localization and stability, human RPE cells were exposed to dextrin conjugated to Oregon green and co-labelled with Lamp-1 (a marker for lysosomes). To assess the effect of GST-conjugate on lipofuscin formation and degradation, cultured RPE cells were either a) fed every two days with photoreceptor outer segments (POS) or b) prepared containing mature lipofuscin granules in the presence or absence of free GST or GST-conjugate for up to 28 days. Lipofuscin levels were determined by FACS and microscopy. Photoxicity was assessed by exposing treated cells to blue light and measuring viability by the MTT assay. For in vivo efficacy and localization studies Oregon-green conjugate was given by intravitreal injection into mice and eyes examined by light and confocal microscopy.
The dextrin-GST conjugate eluted as a single peak and was the only major band by electrophoresis. Over 90% of the polymer was taken up by the lysosomes within RPE cells and remained for up to 24 hours. The enzyme activity of the GST-conjugate was similar to unconjugated GST. Both free and dextrin-conjugated GST reduced the rate of lipofuscin accumulation in RPE cells fed isolated POS by 42% and 65% respectively after 28 days compared to cells treated with POS alone. In cells containing mature lipofuscin, the GST-conjugate led to a greater than 50% decrease in lipofuscin by 28 days. Electron microscopy showed a reduction in the number of lipofuscin granules and a reduced electron density of the remaining lipofuscin granules in dextrin-GST conjugate treated cells. Free GST had no effect on the degradation of lipofuscin. Photoxicity was significantly less in lipofuscin-containing RPE cells treated with GST-conjugate compared to untreated controls. Moreover, intravitreal injection of dextrin-Oregon green conjugate confirmed localization to lysosomes in the mouse retina and no overt retinal damage.
The results from this study confirm that a dextrin-GST conjugate can both inhibit lipofuscin formation and promote degradation of existing lipofuscin. This may offer a novel therapy in lipofuscin-associated diseases such as AMD.
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