Molecular Mechanisms Mediating the eEdocytosis of Biodegradable PLGA Nanoparticles in Rabbit Conjunctival Epithelial Cell Layers

Abstract

Abstract: : Purpose:The uptake of poly (dl-lactide co-glycolide) (PLGA) nanoparticles was shown previously to occur by endocytosis, as evidenced by its punctate distribution pattern under confocal microscopy and its inhibition by cytochalasin D (an endocytosis inhibitor). We sought to identify the role of clathrin and caveolin in the endocytosis of biodegradable PLGA nanoparticles in primary culture of rabbit conjunctival epithelial cells (RCECs). Methods:PLGA (50:50 ratio) nanoparticles (100 nm) containing Texas-red bovine serum albumin (a fluorescent marker, 10.5% w/w) were used. The effect of clathrin inhibitors (hypertonic medium and K+-depletion) and caveolae inhibitors (nystatin and filipin) on nanoparticle uptake was investigated. Western blot and fluorescent confocal microscopy were performed to detect clathrin and caveolin-1 proteins in RCECs. HeLa cells (rich in clathrin) and A431 human epidermoid cells (abundant in caveolin-1) were used as positive controls. A pair of primers was designed (based on conserved regions among different species) to amplify a 180-bp fragment of caveolin-1 gene. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for caveolin-1 was performed using total RNA prepared from freshly isolated rabbit conjunctival epithelial cells for comparison with corneal and tracheal epithelial cells, heart muscle, brain, and HEK293 cells. The RT-PCR products were separated using 2% agarose gel electrophoresis. Results:Nanoparticles uptake was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. In contrast, nystatin and filipin had no effect on nanoparticle uptake in RCECs. Western blot revealed a clathrin band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Caveolin-1 protein was reported to exist in conjunctival tissue, which may be of endothelial cell origin. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb. The absence of caveolin-1 gene in RCECs was confirmed by RT-PCR, as indicated by a 180-bp fragment of the gene. Conclusion:Endocytosis of nanoparticles in RCECs is probably mediated by clathrin-coated and/or other types of vesicles, but not by caveolin-1 vesicles.

Keywords: 365 conjunctiva • 417 gene/expression • 471 microscopy: confocal/tunneling