May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Hyluronan Receptor-Mediated Uptake of Hyaluronic Acid-Chitosan Nanoparticles in Ocular Epithelial Cells
Author Affiliations & Notes
  • L. Contreras
    Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain
  • A. López-García
    Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain
  • M. Calonge
    Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain
    Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER BBN), Valladolid, Spain
  • M. de la Fuente
    Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain
  • B. Seijo
    Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain
  • M. J. Alonso
    Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain
  • Y. Diebold
    Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain
    Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER BBN), Valladolid, Spain
  • Footnotes
    Commercial Relationships  L. Contreras, None; A. López-García, None; M. Calonge, None; M. de la Fuente, None; B. Seijo, None; M.J. Alonso, None; Y. Diebold, None.
  • Footnotes
    Support  FEDER-CICYT MAT2007-64626-C02-01/C02-02, and FPU Scholarship Program, Ministry of Education and Science, and CIBER-BBN, Ministry of Health, Spain.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4785. doi:https://doi.org/
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      L. Contreras, A. López-García, M. Calonge, M. de la Fuente, B. Seijo, M. J. Alonso, Y. Diebold; Hyluronan Receptor-Mediated Uptake of Hyaluronic Acid-Chitosan Nanoparticles in Ocular Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4785. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To determine whether the main hyaluronan receptors, CD44 and RHAMM, are implicated in the cellular uptake of hyaluronic acid and chitosan-based nanoparticles (HA-CS NPs), proposed by our group as drug delivery systems for ocular surface disorders.

Methods: : We first analyzed the expression of hyaluronan receptors in human epithelial cell lines from conjunctiva (IOBA-NHC) and cornea (HCE) and in control tissues from the ocular surface (conjunctiva, limbus and cornea). Cells and tissues were subjected to electrophoresis and Western blotting and immunofluorescence microscopy to detect CD44 and RHAMM. HA-CS NPs conjugated with fluoresceinamine and containing model plasmid DNA were prepared by ionotropic gelation and suspended in HBSS. Cells lines were exposed to 20 µg/ml HA-CS NPs for 1 hour at 37ºC and their cellular uptake was studied over a period of time (1, 6, 24 and 48 hours) using fluorescence microscopy and fluorometry. Experimental groups included: cells incubated with the monoclonal antibody Hermes-1 or with an excess of hyaluronic acid to block the hyaluronan receptors, and control cells. Statistical analysis was the three-way ANOVA.

Results: : Different isoforms of CD44 and RHAMM were detected in IOBA-NHC and HCE cells and in control tissues. Both cell lines and tissues showed higher expression levels of CD44 than RHAMM, as determined by Western blotting and inmunofluorescence. CD44 was uniformly expressed in conjunctival, limbal and corneal epithelia; however, its expression was higher in HCE cells than in IOBA-NHC cells. RHAMM expression was similar in both cell lines, although it was higher in conjunctival epithelium than in the other epithelia. HA-CS NPs were extensively internalized at 37ºC; however, their uptake significantly decreased after blocking hyaluronan receptors, as determined by fluorometry and microscopy. Hyaluronic acid was more effective than Hermes-1 antibody in both cell lines.

Conclusions: : The hyaluronan receptor-mediated uptake may be one of the potential mechanisms to explain HA-CS NPs internalization by ocular surface cells. These findings may have an impact in order to consider these NPs effective drug delivery systems for ocular application.

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