Characterization and Anti-Inflammatory Effects on Periodontal Ligament Cells of Citrus limon-Derived Exosome-like Nanovesicles Under Different Storage Temperatures

<b>Objectives</b>: The purpose of this study is to compare the differences between lemon-derived exosome-like nanovesicles (LELNs) stored at -80 °C, -20 °C, and 4 °C for one month and freshly isolated LELNs, in terms of characterization and anti-inflammatory effects on periodontal ligament cells, aiming to identify suitable storage conditions for LELNs. <b>Methods:</b> Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and micro bicinchoninic acid assay (BCA) were conducted to access the characterization differences. LPS-induced human periodontal ligament cells were used as an in vitro inflammatory model, and the changes in biological functions were examined by qRT-PCR and ELISA. <b>Results:</b> LELNs stored at -80 °C retained the highest particle and protein concentration and showed the least aggregation and heterogeneity in size on TEM images, while the average particle sizes shown by NTA were similar. And LELNs exhibited similar anti-inflammatory effects on periodontal ligament cells after one month of storage at -80 °C, -20 °C, and 4 °C. <b>Conclusions:</b> We found that LELNs can maintain in vitro anti-inflammatory ability when stored at either -80 °C, -20 °C, or 4 °C for one month, while storing at -80 °C maintains the concentration and uniform particle size best.