Paracellular and Transcellular Permeability in Human Corneal Epithelial Cell Culture Model

Abstract

Abstract: : Purpose: To characterize the paracellular and transcellular permeability and esterase activity of recently introduced culture model of corneal epithelium (HCE). Methods: Immortalized human corneal epithelial cells were grown on collagen coated filters under air-lift. Sensitivity of HCE-model to permeant lipophilicity was tested by measuring permeability of 9 beta-blockers. The paracellular pathway was investigated for the size and charge selectivity with 16 polyethylene glycol oligomers (PEG) and 17 cationic amino-PEGs. An effusion–like approach was used to estimate pore sizes and porosity of the paracellular route in HCE membrane. Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity. Drug concentrations were analyzed using HPLC (b -blockers), LC-MS (PEGs), and fluorometry (fluorescein). For comparison, the permeabilities in excised rabbit cornea were determined in vitro. Results: Penetration of b-blockers increased about 15 fold with lipophilicity according to a sigmoidal relationship similarly with excised cornea. Paracellular permeability in HCE-model was slightly higher than in excised rabbit cornea. Based on effusion analysis the HCE model has larger paracellular pores (8.7 Å) but lower pore density (11.9 x 106/cm2) than the excised cornea (7.3 Å and 12.5x106/cm2). Charge selectivity of the membrane decreased the paracellular permeability of amino-PEGs as compared with neutral PEGs. HCE-model had substantial esterase activity (fluorescein diacetate was converted to fluorescein). Conclusions: These data demonstrate that the barrier of HCE-model closely resembles that of the excised rabbit cornea. HCE-model is a promising alternative model of the cornea in ocular drug delivery studies.

Keywords: cornea: epithelium • pharmacology • drug toxicity/drug effects