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K. T. Nguyen, I. Kozak, M. C. Davidson, W. R. Freeman, G. A. Silva; Distribution of Anti-VEGF Molecules in ARPE-19 Cells Using Quantum Dot Labeling. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1861.
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Our goal is to develop ways to visualize the distribution of anti-VEGF drugs, in particular bevacizumab (Avastin), within retinal tissues using chemically functionalized quantum dot nanotechnology. Through this technique, it may be possible to visualize individual molecules of Avastin and to elucidate their distribution within ocular tissue cells.
Human ARPE-19 cells were cultured to confluency and fixed in 4% paraformaldehyde. The cells were then treated and labeled with biotinylated-Avastin conjugated to streptavidin-quantum dots. Quantum dots are fluorescent semi-conductor nanocrystals, which can be functionalized with antibodies and other molecules for targeted labeling. Cells were washed to remove non-bound Avastin and imaged with an Olympus IX81 inverted fluorescent confocal microscope. Avastin is an antibody for vascular endothelial growth factor (VEGF), thus the distribution of Avastin on RPE cells reflects the distribution of VEGF molecules bound to Avastin. The controls include cultures labeled with unconjugated-quantum dots and cultures treated with Avastin labeled with another fluorophore, fluorescein isothiocyanate (FITC).
Quantum-dot labeling provided specific binding and bright fluorescence, yielding a high signal-to-noise ratio. This new method of labeling Avastin on RPE cells shows clear labeling of Avastin bound VEGF molecules. Visualization of VEGF molecules shows distribution throughout the cell excluding the nucleus. Regarding controls, cultures with unconjugated-quantum dots resulted in no fluorescence as expected; cultures treated with Avastin labeled with FITC resulted in similar images to quantum dots except in specificity and background fluorescence.
This is the first use of quantum dot labeling of individual molecules of Avastin. It provides more accurate visualization of these molecules than currently available methods. Using this method, it might be possible to quantify the distribution of VEGF molecules as well as its receptors. Ongoing experiments aim to elucidate the role of Avastin and other anti-VEGF inhibitors in RPE cells and its possible effect on reuptake of fluid during treatments.
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