Stabilization and cellular delivery of chitosan-polyphosphate nanoparticles by incorporation of iron

Chitosan (CS) nanoparticles are typically obtained by complexation with tripolyphosphate (TPP) ions, or more recently using triphosphate group-containing drugs such as adenosine triphosphate (ATP). ATP is an active molecule we aim to deliver in order to restore its depletion in macrophages, when associated with their death leading to plaque rupture in atherosclerotic lesions. Despite high interest in CS nanoparticles for drug delivery, due to the biodegradability of CS and to the ease of the preparation process, these systems tend to readily disintegrate when diluted in physiological media. Some stabilization strategies have been proposed so far but they typically involve the addition of a coating agent or chemical cross-linkers. In this study, we propose the complexation of CS with iron ions prior to nanoparticle formation as a strategy to improve the carrier stability. This can be achieved thanks to the ability of iron to strongly bind both chitosan and phosphate groups. Nanoparticles were obtained from either TPP or ATP and chitosan-iron (CS-Fe) complexes containing 3 to 12% w/w iron. Isothermal titration calorimetry showed that the binding affinity of TPP and ATP to CS-Fe increased with the iron content of CS-Fe complexes. The stability of these nanoparticles in physiological conditions was evaluated by turbidity and by fluorescence fluctuation in real time upon dilution by electrolytes, and revealed an important stabilization effect of CS-Fe compared to CS, increasing with the iron content. Furthermore, in vitro studies on two macrophage cell lines (J774A.1 and THP-1) revealed that ATP uptake is improved consistently with the iron content of CS-Fe/ATP nanoparticles, and correlated to their lower dissociation in biological medium, allowing interesting perspectives for the intracellular delivery of ATP.