Preparation and evaluation of chitosan nanoparticles containing Diphtheria toxoid as new carriers for nasal vaccine delivery in mice

The aim of the present work was to investigate the potential utility of nanoparticles made of chitosan (CS) and also CS chemically modified with polyethylene glycol (CS-PEG) as new vehicles for improving nasal vaccine delivery. For this purpose, diphtheria toxoid (DT) was chosen as a model antigen. DT was entrapped within nanoparticles made of CS of different molecular weight, and also made of CS-PEG, by an ionic cross linking technique. DT-loaded nanoparticles were characterized for their size, surface charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. The resulting nanoparticles had a size, which varied depending on the formulation conditions and on the PEG derivatization, between 100 and 500 nm. They exhibited a positive electrical charge (approx. +40 mV) which was substantially reduced for the PEGgylated CS nanoparticles (approx. +10 mV) and showed and excellent DT loading capacity (loading efficiency between 50-100% depending on the formulation). The results of the in vitro release studies displayed a biphasic release of antigenically active toxoid, the intensity of the first phase being less pronounced for CS-PEG nanoparticles than for CS nanoparticles. Following intranasal administration, DT-loaded nanoparticles elicited an increasing and enhanced humoral immunogenic response (IgG titers), as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) achieved at 70 days post-administration was significantly higher for the DT-loaded CS nanoparticles than for the fluid vaccine. Interestingly, this response was not affected by the CS molecular weight but it was positively influenced by the PEGylation of CS. CS and CS-PEG nanoparticles are promising carriers for nasal immunization with DT.