Internalization and intracellular fate of hyaluronic acid‐coated chitosan nanoparticles in cultured cancer cells growth at physiological pH.

Conventional chemotherapy has been shown to have limited efficacy, due to chemoresistance and treatment-related side effects on healthy tissues. Presently, one of the main challenges for researchers is to develop novel approaches to specifically deliver anticancer drugs on cancer tissue. For this purpose, nanomedicine is deeply investigated to be an efficient new anticancer tool. The membrane receptor CD44 was identified as a potential target of a novel anticancer approach. Indeed, CD44 is largely reported to be involved in the crucial mechanism of tumorigenesis transformation, its overexpression is related to the poor prognosis and it has been identified as a suitable anticancer target. The hyaluronic acid (HA) is an extracellular matrix component that is the main ligand of CD44. This polymer is internalized by the activation of CD44-mediated internalization that involves the endo-lysosomal pathway. Several investigations propose HA to the design of nanocarriers with specific targeting features. Starting with these assumptions, a successful approach might be represented by a new formulation of chitosan/hyaluronic acid nanoparticles (HA//CSNPs). These NPs combine the possibility of HA to assess active targeting of CD44+ cancer cells with CS that thanks to its positive charge might interact and transports the negative charge molecules as therapeutic cargo, such as nucleic acids, and be proposed as a novel anticancer approach. Here, we presented the characterization of a new generation HA//CS NPs that differs from the previous for their stability at physiological pH. We assess the active role of CD44 in the internalization process of our HA//CS NPs in melanoma cancer cells (A375) by cytofluorimetry and confocal microscopy. Furthermore, we identified the NPs kinetics of internalization in A375 cells by FACS analysis of fluorescent HA-Cy3//CS-AF488 NPs, we then quantified the intracellular sorting of the two polymers in order to investigate the intracellular fate of the polymers, testing the hypothesis of CS lysosomal escape, identified as a unique mechanism of protection of the cargo against the lysosomal degradation. Globally, our data increase the knowledge on the HA/CS nanocarriers, and the active role of CD44 on their internalization reinforces the idea that they might represent a promising approach to further develop efficient HA-carrier systems as an anti-cancer tool.