Thiolated polymers have attracted more and more attention for their prominent efflux pump inhibitory properties. The aim of this study was to unravel the P-glycoprotein (P-gp) inhibitory mechanisms mediated by new synthetic thiolated chitosan, chitosan-thioglycolic acid (CS-TGA) and its S-protected derivative (CS-TGA-6MNA). P-gp inhibitory activity was first assessed by determining rhodamine-123 (Rho-123) accumulation in Caco-2 cells, transportation across Caco-2 cell monolayers and across freshly excised rat intestine after exposure to CS-TGA or CS-TGA-6MNA. The results showed that after incubation with CS-TGA or CS-TGA-6MNA, a significant increase in intracellular Rho-123 level was observed in Caco-2 cells, more transportation in the absorptive (AP -> BL) direction and less transportation in the secretory (BL -> AP) direction of Rho-123 was observed in Caco-2 cell monolayers compared with DMEM solution control. In freshly excised rat intestine transportation, more Rho-123 was transported in a concentration-dependent manner. Then the P-gp inhibitory mechanisms were evaluated from four aspects: P-gp expression level, P-gp ATPase activity, intracellular ATP level and plasma membrane fluidity. No obvious changes in P-gp expression or intracellular ATP level were detected after exposure. However, the plasma membrane fluidization decreased and the P-gp ATPase activity reduced, which might contribute to the P-gp inhibitory effect. These features suggested this novel biomaterial might contribute to enhance the oral bioavailability of P-gp substrate drugs, such as cyclosporine A and paclitaxel.
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