Calcium-Containing Squalene Emulsion as a Novel Vaccine Adjuvant Delivery System

Objective: To construct a novel calcium ion–based squalene water-in-oil emulsion, termed calcium ion reverse micelle squalene emulsion (CISE), as a vaccine adjuvant delivery system by exploiting the high affinity of calcium ions for phosphate groups and biomacromolecules. Methods: CISE was prepared using an ultrasonic emulsification method and characterized by laser particle size analysis and transmission electron microscopy (TEM). Its adjuvant efficacy and biocompatibility were evaluated through both in vitro and in vivo experiments. Results: CISE has a particle size of approximately 124.7 nm and exhibits good dispersibility. In vitro experiments show that antigen presenting cells (APCs) efficiently internalize CISE primarily through caveolae mediated endocytosis. CISE promotes the maturation and activation of bone marrow derived dendritic cells (BMDCs), accompanied by elevated production of reactive oxygen species (ROS) and enhanced peroxisomal activity. Notably, CISE possesses prominent lysosomal escape capacity within APCs. After two rounds of subcutaneous immunization in mice, CISE induced high levels of IgG antibodies and elicited a relatively balanced Th1/Th2 immune response. Following pulmonary mucosal immunization, CISE not only triggered robust IgG production but also induced high levels of IgA antibodies. Conclusions: When loaded with a model antigen, CISE effectively induces humoral, cellular, and mucosal immunity, demonstrating significant potential as a vaccine adjuvant.