Maria de la Fuente1, Begoña Seijo, Maria J Alonso. 1. Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain.
Abstract
PURPOSE: Gene therapy offers a promising alternative for the treatment of ocular diseases. However, the implementation of this type of therapy is actually hampered by the lack of an efficient ocular gene delivery carrier. The main objective of the present work was to assess the effectiveness and investigate the mechanism of action of a new type of nanoparticle made of two bioadhesive polysaccharides, hyaluronic acid (HA) and chitosan (CS), intended for the delivery of genes to the cornea and conjunctiva. METHODS: The nanoparticles were obtained by a very mild ionotropic gelation technique. They were loaded with either the model plasmid pEGFP or pbeta-gal. Transfection and toxicological studies were conducted in human corneal epithelial (HCE) and normal human conjunctival (IOBA-NHC) cell lines. The mechanism of internalization of the nanoparticles by the corneal and conjunctival cells was investigated by using fluorescence confocal microscopy. RESULTS: The nanoparticles had a size in the range of 100 to 235 nm and a zeta-potential of -30 to +28 mV. The results of the transfection studies showed that HA-CS nanoparticles were able to provide high transfection levels (up to 15% of cells transfected), without affecting cell viability. The confocal images indicated that HA-CS nanoparticles were internalized by fluid endocytosis and that this endocytic process was mediated by the hyaluronan receptor CD44. CONCLUSIONS: The results give evidence of the potential of HA-CS nanoparticles for the targeting and further transfer of genes to the ocular surface.
PURPOSE: Gene therapy offers a promising alternative for the treatment of ocular diseases. However, the implementation of this type of therapy is actually hampered by the lack of an efficient ocular gene delivery carrier. The main objective of the present work was to assess the effectiveness and investigate the mechanism of action of a new type of nanoparticle made of two bioadhesive polysaccharides, hyaluronic acid (HA) and chitosan (CS), intended for the delivery of genes to the cornea and conjunctiva. METHODS: The nanoparticles were obtained by a very mild ionotropic gelation technique. They were loaded with either the model plasmid pEGFP or pbeta-gal. Transfection and toxicological studies were conducted in humancorneal epithelial (HCE) and normal human conjunctival (IOBA-NHC) cell lines. The mechanism of internalization of the nanoparticles by the corneal and conjunctival cells was investigated by using fluorescence confocal microscopy. RESULTS: The nanoparticles had a size in the range of 100 to 235 nm and a zeta-potential of -30 to +28 mV. The results of the transfection studies showed that HA-CS nanoparticles were able to provide high transfection levels (up to 15% of cells transfected), without affecting cell viability. The confocal images indicated that HA-CS nanoparticles were internalized by fluid endocytosis and that this endocytic process was mediated by the hyaluronan receptor CD44. CONCLUSIONS: The results give evidence of the potential of HA-CS nanoparticles for the targeting and further transfer of genes to the ocular surface.