Enhancing the in vivo efficacy of anthrax vaccine using trimethylchitosan covalently coated chitosomes in a single-step microfluidic synthesis.

Liposomal vaccines have been developed extensively in recent years, and considerable progress has been made in the prevention of infectious diseases. Quaternary chitosan-coated liposomes were used in the present study. Liposomes coated with TMC (namely chitosomes) were synthesized in a single microfluidic step in which liposomes were formed and coated with trimethylchitosan (TMC) via covalent and/or hydrogen bonds in situ. The endocytosis of chitosomes by JAWS II dendritic cells was detected using flow cytometry. Chitosomes covalently coated with TMC were more efficiently internalized than uncoated liposomes, showed lower cytotoxicity than those coated via hydrogen bonds, and could significantly lower the cytotoxicity via protein mixing. In a BALB/c model, covalent modification of chitosomes improved the efficiency of anti-PA IgG induction via anthrax vaccine adsorbed (AnV), compared with those with hydrogen bonds and those with AnV only, where humoral immunity is dominant. These results suggest that covalently TMC-coated chitosomes serve as a promising delivery system for proteins and that they can be reproducibly obtained via rapid, cost-efficient, and automated methods with few scale-up barriers; meanwhile, their versatile practicability favors commercialization. STATEMENT OF SIGNIFICANCE: In this study, we proved that liposomes coated with TMC (namely chitosomes) via covalent bonds induced better uptake efficiency, stronger in vivo immune responses, and lower cytotoxicity than those coated via hydrogen bonds. The endocytosis efficiency and cytotoxicity of chitosomes were positively correlated with the concentration of the TMC coating. Additionally, this invention provides a facile single-step microfluidic process for the rapid, low-cost, and high-quality preparation of an innovative high-performance chitosomal delivery system that is convenient and easy to scale up. Based on these benefits, the current disclosure provides an alternative platform for the development of new chitosomal drugs.