Cell internalization study of hybrid chitosan-heparin-PLGA nanocarriers on Caco-2 cells

Purpose: Oral delivery of Heparin, the most potent anticoagulant drug, could have a great clinical impact, offering undoubted advantages over injectable formulations. The aim of this work was to evaluate the ability of hybrid Chitosan-Heparin-PLGA nanocarriers to be taken up by CaCo-2 cells, a model of mature enterocytes of the absorbent epithelium of intestine. Methods: Firstly, Chitosan/Heparin nanocoacervates were prepared by polyelectrolyte complexation method; subsequently, the coacervates were coated with PLGA to be stabilized at intestinal pH, resulting in hybrid Chitosan-Heparin-PLGA nanocarriers. Free Chitosan/Heparin nanocoacervates and hybrid nanoparticles were characterized regarding size, z-potential, morphology and Heparin release in gastric and intestinal simulated media. The influence of the size and of the time contact with cells on the ability of the carriers to be internalized was studied on Caco-2 cell monolayers by using fluorescence microscopy. In order to visualize the carriers, FITC-Chitosan and Rodamine- PLGA were employed. Results: It was found that in the case of free coacervates (from 300 to 500 nm) the smaller the size, the higher was the extent of cell internalization, regardless of the time contact with cells (4 or 18 h). On the contrary, in the case of hybrid Chitosan-Heparin-PLGA nanocarriers, showing an increased size (until 700 nm) due to the polymeric coating, the cell internalization increased according to the contact-time with cells. Conclusion: The size of nanocarriers greatly influences the cell internalization rate in CaCo-2 cells. Although hybrid systems showed a low internalization rate, being stable in the intestinal environment unlike the free coacervates, they seem a promising approach to be further investigated for the oral absorption of Heparin.