Effects of high molecular weight n-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride on the barrier integrity of Caco-2 monolayer

Abstract

Development of poorly soluble and unstable peptide drugs using drug delivery system has been introduced to improve drug bioavailability via oral administration. Among the oral drug carriers interested in synthetic polymers, 600-HPTChC is the modified chitosan with quaternization process to improve solubility without any effect on the mucoadhesive property of core chitosan. However, the biocompatibility of 600-HPTChC on human intestine and its mechanism on paracellular and transcellular transport remained to be unknown. The purposes of this study were therefore to determine the effects of 600-HPTChC on intestinal cells in aspects of cell proliferation and cell differentiation and to examine the potential effects of 600-HPTChC on improvement of drug bioavailability through paracellular and transcellular pathway using Caco-2 cells as an in vitro intestinal model.

The results demonstrated that lower degree of substitution of 600-HPTChC caused lower cytotoxic and anti-proliferative effect on Caco-2 cells comparing to the higher degree of substitution. In addition, anti-proliferative effect of 600-HPTChC might be involved by cell cycle disturbance further causing differentiation delay of the Caco-2 cells after the continuous exposure for 9 days. However, 4 h/day exposure over 9 days, more realistically mimics the daily intestinal exposure, could attenuate the effect of 600-HPTChC on intestinal differentiation. In paracellular pathway, 600-HPTChC was much more efficient than chitosan in both aspects of opening rate and time recovery of tight junction protein. The results also suggested that the mechanism behind the tight junction opening might result from the direct interaction between 600-HPTChC and tight junction protein rather than via intracellular signaling investigated. In transcellular pathway, 600-HPTChC was able to increase the drug absorption by P-gp inhibition without any change in the expression level. Immunofluorescent images indicated that 600-HPTChC could directly bind to P-gp on plasma membrane. Furthermore, the results of P-gp ATPase assay demonstrated that 600-HPTChC acts as the stimulator of P-gp ATPase that might relate to conformation change of P-gp epitope. Overall data support the usefulness of 600-HPTChC as a drug carrier in development of the oral drug delivery system.