Extracellular vesicles protect glucuronidase model enzymes during freeze-drying

Abstract

Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including −80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, β-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.

Biocompatible bacteria-derived vesicles show inherent antimicrobial activity

“…Interestingly, a major size drift was observed when storing these OMVs at 4 °C (Figure 2d). Until now, there are very little reports on a standardized and preferred method for storing OMVs and EVs yet although it is essential for advanced studies [42,47] and storage of vesicles at -80 °C is recommended [48]. Our results indicate that freeze-drying is a valid alternative for storing OMVs as it was also shown in literature [42,49].…”

“…Each OMV sample was stored in PBS and at 4 °C, -20 °C, -80 °C and freeze dried to evaluate their stability [42]. Aliquots of 100 µL of each sample were kept in MaxyClear microtubes (Axygen) to avoid vesicles absorption to the plastic surface of the tubes.…”

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses

“…In this context, cryoprotectants such as trehalose, already used for labile proteins, vaccines, and liposomes, appear to have a positive impact on EVs . Additionally, Frank et al reported that freeze‐drying does not significantly impact the size and particle number of MSC‐derived EVs and that, upon addition of cryoprotecting sugars, the enzymes in the lyophilized EVs have comparable activity to the ones in EVs stored at −80 °C.…”

A sustainable amperometric biosensor for the analysis of ascorbic, benzoic, gallic and kojic acids through catechol detection. Innovation and signal processing

“…In recent years, it has been observed that stability and final activity of freeze-dried materials critically depend on how the entire approach is performed. [22][23][24][25] Essentially, this is based on different freezing and drying stresses, such as solute concentration, formation of ice crystals, pH changes and others, that are prone to denature proteins in the material to various degrees. 22 Thus, stabilizers are often required in a protein formulation to protect its stability in the final processed material.…”