Conjunctival epithelial uptake of biodegradable nanoparticles: Mechanism, intracellular distribution, and absorption enhancement

This investigation was prompted by the potential application of biodegradable PLGA nanoparticles as ocular carriers for protein and drug delivery. The overall goal is to improve the mechanistic understanding of nanoparticle transport and trafficking within the conjunctival epithelium to facilitate the design of successful nanoparticle systems capable of enhancing the delivery of proteins and drugs to the conjunctiva and other intraocular tissues. Our data provided evidence for the endocytosis of PLGA nanoparticles in rabbit conjunctival epithelial cells (RCECs), as demonstrated by the uptake inhibition in the presence of microfilament inhibitors and metabolic poisons, the vesicular uptake pattern seen under confocal microscopy, and the stimulatory effect on endocytosis of the fluid phase marker, Lucifer yellow.; Evidence is presented for the mechanism of PLGA nanoparticle uptake in RCECs. Based on clathrin knockout studies, we demonstrated lack of a direct involvement of clathrin heavy chain in endocytosis of PLGA nanoparticles. However, inhibition studies using pharmacological treatments (hypertonicity and intracellular K+ depletion), the partial colocalization of clathrin staining seen under confocal microscopy, and the incomplete knockout of clathrin protein suggest a minor involvement of clathrin in nanoparticle endocytosis. We have provided the first evidence for the expression of clathrin heavy chain at the protein and gene levels in rabbit conjunctival epithelial cells, whereas caveolin-1 expression was not detected. Since macropinosomes are involved in endocytosis of larger particles (>500 nm), our findings implicate mainly non-coated vesicles and clathrin-coated vesicles partially in the internalization of PLGA nanoparticles in RCECs.; Immunofluorescence staining studies delineated the intracellular distribution and trafficking behavior of PLGA nanoparticles following endocytosis. Nanoparticles were shown to escape endosomal trafficking, reside for a short while in the lysosomes, and localize to the Golgi compartment during their trafficking in RCECs. We have also provided evidence for the utility of using PLGA nanoparticles for protein drug delivery and controlled drug delivery in the conjunctival epithelium, and probably to other intraocular tissues. Finally, the feasibility of using lectins from Solanum tuberosum (potato) to augment nanoparticle absorption in RCECs has been highlighted and shown to be efficacious and safe of ocular use as drug carriers.