Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles.
OBJECTIVE
To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers.


METHODS
Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts.


RESULTS
Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer.


CONCLUSIONS
Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.
Ma
Zengshuan
Z
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543.
Lim
Lee-Yong
LY
D018938:Caco-2 Cells; D002686:Chitin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D048271:Chitosan; D006801:Humans; D007328:Insulin / Q000493:pharmacokinetics*; D036103:Nanotechnology / Q000379:methods*
D007328:Insulin; D002686:Chitin; D048271:Chitosan