Agarose-grafting-hyaluronan (Ag-g-HA) has been proved to be a potential carrier for oral insulin, but it needs to clarify that insulin loaded in Ag-g-HA microparticle is effectively transported across epithelial layer. In this paper, Caco-2 cell monolayer was used for imitation of epithelial layer in small intestine to evaluate Ag-g-HA copolymer enhanced insulin transportation crossing epithelial layer. Ag-g-HA/insulin polyelectrolyte complexes were formed via electrostatic interaction at pH 3-5.4 and could spontaneously form microparticles with average diameter of 2 mu m. Caco-2 cells in transwell dish were attached in tight monolayer after 21 d proliferation, and some features of Caco-2 cell monolayer, such as trans-epithelial electrical resistance (TEER) value being over 400 Omega/cm(2), alkaline phosphatase (ALP) activity being 0.7184 +/- 0.095, and apparent permeability coefficient value (P(app)) of propranonol being 1 x 10(-6) cm/s, were similar to those of epithelial layer in small intestine. These results revealed that Caco-2 cell monolayer was suitable to be a model of small intestine epithelium layer for imitation of insulin crossing small intestine. Papp of insulin crossing Caco-2 monolayer reached 0.592 +/- 0.067 x 10(-6) cm/s, which was significantly greater than the control groups. This meant Ag-g-HA as insulin carrier significantly promoted insulin crossing Caco-2 cell monolayer. These findings demonstrate that Caco-2 cell monolayer is a suitable model for drug transportation crossing epithelial layer, and Ag-g-HA has a promise as oral insulin carrier. (c) 2010 Elsevier B.V. All rights reserved.