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Nuria Genicio, Juan Gallo, Marc Dziasko, Nicholas J Long, Alex J Shortt; Biofunctionalized heavy metal free quantum dots (QD) as a tool for tracking transplanted limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5508.
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Cultured limbal epithelial stem cells (LESC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to use advances in QD technology to develop a novel tool for tracking LESC in human clinical trials.
A novel heavy metal free QD (HMF-QD) composed of an InP core, ZnS shell and a polyethylene glycol (PEG) coating was functionalized by conjugation with glucose. The effect of glucose conjugation on HMF-QD uptake was investigated in a human corneal epithelial cell line (HCE-T) and in primary human limbal epithelium (HLE). Uptake was evaluated by flow cytometry, photoluminescence, confocal microscopy and electron microscopy. A range of HMF-QD concentrations and exposure times were investigated to determine the optimum labelling protocol. Cytotoxicity was evaluated using flow cytometry with propidium iodide staining. The duration and intensity of labelling was examined over a 28-day period using confocal microscopy. Heavy metal containing Cd/Se QDs were used as a control.
HMF-QD coated with PEG alone demonstrated poor uptake by both HCE-T and primary HLE. Bioconjungation with glucose (Glc-HMF-QDs) dramatically increased uptake. Evaluation of loading concentration and exposure time showed a dose and exposure dependent increase in the percentage of positive cells (flow cytometry) the overall fluorescence of cell cultures (photoluminescence). The optimal labelling protocol was a 2-hour incubation with 50µg/ml Glc-HMF-QDs which resulted in >90% of cells being labelled and a strong fluorescence signal at 595nm that was visible by confocal microscopy. This protocol resulted in cell viability of 97% whereas increasing the incubation time to 24 hours caused a dose dependent decrease in viability. Electron microscopy showed the intracellular distribution of Glc-HMF-QDs within the cytoplasm. HCE-T labelled with Glc-HMF-QDs were observed in culture for up to 28 days and a strong signal was detectable throughout. Controls labelled with heavy metal containing QDs (Glc-CdSe/ZnS) showed death of all cells by day 6.
Glc-HMF-QDs are a safe and effective tool for labelling HEC-T and HLE cells. To date we have confirmed their efficacy in-vitro. Future work will evaluate their usefulness in in-vivo models of LESC transplantation.
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