title: Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.
abstract: OBJECTIVE
  Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus.
  
  
  METHODS
  HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification.
  
  
  RESULTS
  There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls.
  
  
  CONCLUSIONS
  HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.
authors.affiliation: Ocular Surface Group-IOBA, University of Valladolid, Paseo de Belén 17, Valladolid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Contreras-Ruiz
authors.forename: María
authors.initials: M
authors.lastname: de la Fuente
authors.forename: Jenny E
authors.initials: JE
authors.lastname: Párraga
authors.forename: Antonio
authors.initials: A
authors.lastname: López-García
authors.forename: Itziar
authors.initials: I
authors.lastname: Fernández
authors.forename: Begoña
authors.initials: B
authors.lastname: Seijo
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sánchez
authors.forename: Margarita
authors.initials: M
authors.lastname: Calonge
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Diebold
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D051242:Caveolin 1 / Q000737:chemistry; D002470:Cell Survival; D002478:Cells, Cultured / Q000187:drug effects; D048271:Chitosan / Q000737:chemistry*; D004705:Endocytosis; D005123:Eye / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D006820:Hyaluronic Acid / Q000737:chemistry*; D008247:Lysosomes / Q000737:chemistry; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D013696:Temperature
chemical_list: D051242:Caveolin 1; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D048271:Chitosan; D000469:Alkaline Phosphatase