Drug delivery through the human respiratory tract is a promising field under investigation. A growing effort is focused on developing innovative delivery systems able to escape the clearance mechanisms of the respiratory tract, to improve molecules bioavailability, their absorption and their therapeutic efficacy, in the respiratory mucosa. In this context, the aim of this study was to evaluate the potential of polysaccharide cationic porous nanoparticles (NP+) as airway vectors for proteins. NP+ are successfully used as mucosal vectors in vivo, in many applications, including vaccination, allergy, cancer therapy and drug delivery. However, the mechanisms of NP+ interaction with airway epithelial cells remain poorly understood. We investigated the endocytosis, the exocytosis and the intracellular localization of NP+ in human bronchial epithelial cells. We assessed their toxicity on these cells, and particularly their cyto- and genotoxicity. Finally, we studied and characterized the mechanisms of intracellular delivery of proteins by these nanoparticles, and the influence of their inner composition, on these mechanisms. Our results showed a rapid uptake of NP+ via the clathrin endocytosis pathway, and a significant exocytosis via a cholesterol-dependent mechanism. Moreover, NP+ were located in clathrin vesicles, early endosomes but not in late endosomes nor lysosomes. Interestingly, these nanoparticles quantitatively associated proteins and increased their intracellular delivery, while protecting them from enzymatic degradation at physiological pH. Moreover, the presence of anionic lipids in their inner structure significantly influences their interaction with cells and the mechanisms of intracellular delivery. Finally, toxicity studies show no genotoxicity or cytotoxicity of these nanoparticles at concentrations below 326μg/cm². However, these concentrations are very high and hardly realistic in vivo. In summary, NP+ are not toxic to airway epithelial cells, they strongly interact with these cells and significantly increase protein delivery. This work highlights the importance of developing this type of nanoparticles to deliver molecules via the human respiratory tract.
