Pasteurization and lyophilization affect membrane proteins of pomegranate-derived nanovesicles reducing their functional properties and cellular uptake

Plant-derived nanovesicles (PDNVs) are promising therapeutic agents, valued for their bioactive compound content and as drug delivery systems. However, the effects of industrial food processing techniques on PDNVs remain underexplored. This study evaluated the impact of lyophilization and pasteurization on the properties and uptake efficiency of pomegranate-derived nanovesicles ( Pg NVs). Pg NVs were isolated from pomegranate juice using tangential flow filtration and size exclusion chromatography. Treated Pg NVs were analyzed via transmission electron microscopy, nanoparticle tracking analysis, and LC-MS/MS, assessing their functional properties and cellular uptake. High-purity Pg NVs were obtained, although both treatments reduced their yield. Pre-treated Pg NVs displayed diminished anti-inflammatory and wound-healing capacities, though antioxidant activity remained unaffected. Treated Pg NVs also exhibited lower internalization by human macrophages compared to untreated Pg NVs. Proteomic analyses revealed that the damage of membrane proteins, such as Tetraspanin-8, might be responsible for these effects. Our findings emphasize the need of optimizing processing to preserve PDNV therapeutic potential. • Pasteurization and lyophilization lower the yield and purity of Pg -derived nanovesicles. • Industrial processing alters the proteomic content of Pg NVs affecting membrane proteins. • Pasteurized and lyophilized Pg NVs show lower cell uptake than untreated Pg NVs. • Processed juice nanovesicles retain antioxidant effects but lose anti-inflammatory activity.