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Y. Diebold, A. Enríquez de Salamanca, M. Jarrín, S. Callejo, A. Vila, M.J. Alonso; Uptake of Bioadhesive Nanoparticles in a Normal Human Conjunctiva (NHC) Epithelial Cell Line . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3785.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously demonstrated that chitosan nanoparticles (CSNs) enter conjunctival epithelial cells in vitro with no damage to the cells. This study is aimed at determining whether CSNs cross the plasma membrane by an active transport mechanism (transcellular route) and/or by the paracellular route. Methods: CSNs were prepared according to an ionic gelation process and made fluorescent by associating FITC-BSA to them. The nanometric size of CSNs was 300 nm in average, and their zeta potential was +35 mV. An epithelial cell line from normal human conjunctiva (NHC) was exposed to 0.5 mg/ml FITC-CSNs in PBS for 15 and 30 min, and 1 and 2 h at 37oC and 4oC to investigate: i) time-dependent CSNs uptake, ii) the effect of temperature on the cellular uptake, and iii) the effect of 100 mM sodium azide treatment. Intracellular presence of CSNs was examined by confocal microscopy. Moreover, after incubation, cell-associated fluorescence was determined by fluorescence spectroscopy in cell lysates. In addition, double-labeling with TRITC-phalloidin and FITC-ZO-1 or occludin and visualization by confocal microscopy was performed to study tight junction disruption. Results: CSNs uptake by NHC cells was observed for all time-points. Uptake was significantly reduced under conditions that blocked active transport processes (4oC and the addition of sodium azide). Moreover, no differences were observed in phalloidin, ZO-1, and occludin immunodetection in NHC cells after exposure to CSNs at the different times. These data are compatible with an active internalization process. Conclusions: The results suggest that CSNs enter conjunctival cells by an active transport rather than by a passive mechanism. The fact that CSNs can actively penetrate cells adds further support to the interest of these bioadhesive, colloidal systems as sustained drug delivery systems for the ocular surface. Support: FEDER-CICYT MAT2000-0509-C02-02 and Junta CyL VA051/01
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