Chitosan (CS) is a potential paracellular permeation enhancer for trans-epithelial drug delivery. It has been investigated as an absorption enhancer for facilitating the oral bioavailability of hydrophilic macromolecules in the small intestine. As is well known, CS can transiently open the tight junctions (TJs) between epithelial cells, thus enhancing the paracellular permeability. However, the mechanism of TJ disruption by CS has remained ambiguous. The aim of this study was to investigate, on molecular levels, the effect of CS on TJ integrity in Caco-2 cells. As Caco-2 cells grown to confluence, polarized monolayers spontaneously differentiate to form columnar cell monolayers. Thus, they have been widely used as a representative in vitro model of the small intestine for the evaluation of trans-epithelial drug absorption.
In study I, the observed changes in transepithelial-electrical-resistance measurements and the staining patterns of the monolayer Caco-2 cells demonstrated that CS can transiently and reversibly open the TJs between cells, thus enhancing the paracellular permeability. TJ ultra-structures examined by transmission electron microscopy support the concept that CS did induce transient opening of TJs. We then assessed TJ disruption at the gene and protein expression levels. Our data indicate that exposure to CS followed by recovery resulted in a significant increase in claudin-4 (Cldn4) gene transcription. Additionally, CS treatment induced redistribution of the TJ protein CLDN4 intracellularly following by its degradation in lysosomes, which represented an important contributing factor in TJ weakening, leading to the opening of TJs. The recovery of TJ after CS disruption required CLDN4 protein synthesis. These results suggest that CS regulates TJs by inducing changes in transmembrane CLDN4 protein.
Next, the signaling mechanism that is related to the effect of CS on TJs was further elucidated. In study II, we revealed the potential transduction cascade of TJ opening in Caco-2 cell monolayers subsequent to CS exposure. Experimental results indicated that activation of integrin receptors on cell membranes significantly contributes to CS-mediated TJ disruption, initiating the cascade of TJ opening. Additionally, treatment of Caco-2 cell monolayers with CS led to the clustering of integrins along the cell border, phosphorylation of FAK and Src tyrosine kinases, and results in the regulation of TJ permeability via the redistribution of TJ protein CLDN4 from the cell membrane to the cytosol.
Moreover, although it is well known that the ability of CS to enhance epithelial permeability is in a pH-dependent manner, its underlying molecular mechanism has remained unclear. In study III, the molecular mechanistic insight into the effect of CS on TJ disruption at different pH environments was explored. The experimental results revealed that the direct interaction between CS and integrin V3 on cell surfaces has a crucial role in CS-induced TJ opening, an indication of receptor activation. The mechanism of action appeared to be the electrostatic interaction between the positively-charged CS and the negatively-charged integrin V3. This electrostatic interaction led to the conformation change of integrin V3 and its clustering along the cell border, F-actin reorganization, and CLDN4 down-regulation, eventually resulting in the disruption of TJs and an increase in paracellular permeability. The above observations were all in a pH-dependent manner. As pH increased, CS became less positively charged, thereby losing its capability to interact with integrin V3 and failing to induce the TJ opening. These consequences might help to better understand the molecular mechanism of TJ opening mediated by CS, thereby facilitating the use of CS for trans-epithelial drug delivery.

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