CHIVAX 2.1-Loaded Chitosan Nanoparticles as Intranasal Vaccine Candidates for COVID-19: Development and Murine Safety Assessment

<b>Background/Objectives:</b> Innovative intranasal delivery systems have emerged as a strategy to overcome the limitations of conventional COVID-19 vaccines, including suboptimal mucosal immunity, limited antigen retention, and vaccine hesitancy. This study aimed to evaluate physicochemical properties and murine safety of a novel COVID-19 intranasal vaccine candidate based on CHIVAX 2.1 (CVX)-loaded chitosan nanoparticles (CNPs). <b>Methods:</b> The CVX recombinant protein was encapsulated into CNPs using the ionic gelation method. The nanoparticles were characterized by their physicochemical properties (mean size, zeta potential, morphology, and encapsulation efficiency) and spectroscopic profiles. Mucin adsorption and in vitro release profiles in simulated nasal fluid were also assessed. In vivo compatibility was evaluated through histopathological analysis of tissues in male C-57BL/6J mice following intranasal administration. <b>Results:</b> CNPs exhibited controlled size distribution (38.5-542.5 nm) and high encapsulation efficiency (65.4-92.2%). Zeta potential values supported colloidal stability. TEM analysis confirmed spherical morphology and successful CVX encapsulation, and immunogenic integrity was also demonstrated. Mucin adsorption analysis demonstrated effective nasal retention, particularly in particles ≈90 nm. In vitro release studies revealed a biphasic protein profile, where ≈80% of the recombinant protein was released within 2 h. Importantly, histopathological analyses and weight monitoring of intranasally immunized mice revealed no signs of adverse effects related to toxicity. <b>Conclusions:</b> The ionic gelation encapsulation process preserved the physical and immunological integrity of CVX antigen. Furthermore, the intranasal administration of the CVX-loaded CNPs demonstrated a favorable safety profile in vivo. These findings support the potential of the CVX intranasal vaccine formulation for further immunogenicity studies, with no apparent biosafety concerns.