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bioRxiv · 10.1101/2025.09.13.675998

Impact of storage on the stability and the protective effect of extracellular vesicles released by Candida albicans

Honorato, L.·Bonilla, J. J. A.·Valdez, A. F.·Reis, F.·Kornetz, J.·Sabino, A. L. R. d. N.·Rodrigues, M.·Nosanchuk, J. D.·Nimrichter, L.
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Abstract

Extracellular vesicles (EVs) released by Candida albicans are multi-antigenic compartments considered as promising prototypes for vaccine development. However, their stability, appropriate storage and handling conditions are largely unexplored, which raises questions related to their biotechnological applicability. Here, we evaluated the physical and functional stability of C. albicans EVs under long-term storage. Furthermore, we conducted a comparative analysis of these properties in C. albicans EVs obtained through three commonly utilized isolation protocols documented in the literature. After identifying the most efficient isolation method for optimal yield, we devised a potential quality control for EVs isolation based on protein and sterol ratio. Subsequently, we investigated the impact of drying EVs using vacuum centrifugation at room temperature or -4 {degrees}C and the effect of freeze-thaw cycles in EVs stability. Transmission electron microscopy (TEM) revealed that EVs maintained morphological stability after long-term (up to 4 years) storage at -80 {degrees}C as well as storage at room temperature, 4 {degrees}C and -20 {degrees}C for 7 days with or without vacuum centrifugation, with a tendency of higher recovery when lower temperature is used. Remarkably, all of the C. albicans EVs suspensions maintained their biological properties as demonstrated by their ability to protect Galleria mellonella against C. albicans infection. However, the number of freeze-thaw cycles significantly impacted on the protective effect of the EVs. Overall, our findings demonstrate that C. albicans EVs maintain notable morphological and biological stability of under several conditions, enabling their efficient and reproducible utilization in research and potentially as therapeutic agents. ImportanceExtracellular vesicles (EVs) released by Candida albicans are promising vaccine prototypes due to their multi-antigenic nature. However, their storage and handling conditions are not well understood, raising concerns about their biotechnological use. This study evaluated the long-term physical and functional stability of C. albicans EVs. We compared three isolation methods to identify the most effective one and suggested a quality control measure based on protein and sterol ratios. We also examined the effects of vacuum drying and freeze-thaw cycles on EV stability. Our findings show that C. albicans EVs maintain their biological function after long-term storage at -80 {degrees}C and under various conditions. Notably, their protective effect in an insect model was reduced though repeated freeze-thaw cycles. This research provides valuable insights for the efficient use of these vesicles in future studies.

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Honorato, L., Bonilla, J. J. A., Valdez, A. F., Reis, F., Kornetz, J., Sabino, A. L. R. d. N., Rodrigues, M., Nosanchuk, J. D., Nimrichter, L.. 2025-09-15. Impact of storage on the stability and the protective effect of extracellular vesicles released by Candida albicans. https://doi.org/10.1101/2025.09.13.675998

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