Intracellular Trafficking of Hyaluronic Acid-Chitosan Nanoparticles in Ocular Epithelial Cells

Abstract

Purpose: : To study the intracellular trafficking of previously tested in vitro and in vivo nanoparticulate drug delivery systems in epithelial cells from the ocular surface.

Methods: : Hyaluronic acid and chitosan-based nanoparticles (HA-CS NPs) containing plasmid DNA as a marker molecule were prepared by ionotropic gelation, conjugated with a fluorescence marker and suspended in HBSS. Epithelial cell lines from human ocular surface tissues (IOBA-NHC from conjunctiva, and HCE from cornea) were exposed to HA-CS NPs for 1 hour at 37ºC. Intracellular trafficking of HA-CS NPs was monitored after 1, 6, 24 and 48 hours. The effect of three different metabolic inhibitors on HA-CS NPs uptake was studied: 1) 100 mM sodium azide; 2) 15 µM colchicine; and 3) temperature (4ºC vs. 37ºC). LysoSensor® and LysoTracker® were used to identify inactive and activated lysosomes, respectively. Z-stacks were done to confirm intracellular location of NPs. Three-way ANOVA was performed for statistical analysis.

Results: : A sustained intracellular localization of plasmid-loaded NPs was observed in both corneal and conjunctival cell lines, as determined by fluorescence microscopy. All three inhibitors reduced HA-CS NPs uptake. However, metabolic inhibition by temperature had a significant effect when compared to that what produced by cell incubation with azide or colchicine.Nanoparticle-associated fluorescence diminished dramatically at 48 hours after exposure, whereas lysosome-associated fluorescence increased, indicating active nanoparticle degradation by lysosomes. Weak plasmid-associated fluorescence was localized in the nucleus or in the perinuclear region after 24 and 48 hours.

Conclusions: : Tested HA-CS NPs were taken up and most of them metabolized by ocular surface epithelial cells in vitro in 48 hours. Further experiments are warranted to determine if the studied nanoparticulated systems effectively deliver plasmid DNA in a sustained way to ocular cells.

Keywords: gene transfer/gene therapy • conjunctiva • cornea: epithelium