PURPOSE: The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. METHODS: Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. RESULTS: Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having Km and Vmax of 3.84 microM and 58.14 microg/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K+ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37 degrees C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37 degrees C. CONCLUSIONS: Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.
PURPOSE: The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. METHODS: Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. RESULTS: Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having Km and Vmax of 3.84 microM and 58.14 microg/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K+ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37 degrees C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37 degrees C. CONCLUSIONS: Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.
Authors: M Malatesta; V Galimberti; B Cisterna; M Costanzo; M Biggiogera; C Zancanaro Journal: Histochem Cell Biol Date: 2013-12-20 Impact factor: 4.304