Fabrication of immune-enhancing vesicles from reassembled yeast vacuolar membranes

Yeast vacuoles have recently emerged as promising bio-nanomaterials for drug delivery, offering improved stability and efficacy compared with traditional synthetic systems. Their membranes share structural and compositional similarities with those of mammalian cells, offering excellent biocompatibility and potential for efficient cellular interaction. These unique properties make reassembled vacuoles (ReV) attractive candidates for developing safe and effective therapeutic delivery platforms. This study optimized the vacuole reassembly process to enhance the performance of drug delivery. We compared two methods: Method 1, using conventional long-duration sonication and filtration, and Method 2, featuring an optimized 5-minute sonication without filtration. Reassembled vacuoles produced by method 1 (ReV_Mtd1), induced moderate TLR2 expression, suggesting mild immune priming without significant activation of inflammatory cytokines. In contrast, Reassembled vacuoles produced by method 2 (ReV_Mtd2) demonstrated superior immune activation, showing a dose-dependent upregulation of iNOS and TLR2. Additionally, ReV_Mtd2 achieved an encapsulation efficiency of 12.9 % for daunorubicin (DNR), comparable to native vacuoles, and maintained structural stability over a 12-month period. These findings highlight the potential of ReV_Mtd2 as a robust, biocompatible, and efficient drug delivery system, offering enhanced therapeutic performance and long-term stability.