Ginger-Derived Exosome-Like Nanoparticles: The Effect of Extraction Methods on Metabolites and in vitro Anti-Lung Cancer Activity.

Introduction: In recent years, plant-derived exosome-like nanoparticles (PELNs) have attracted extensive attention. Among them, Ginger-derived exosome-like nanoparticles (GELNs) represent the most extensively studied category, demonstrating a wide spectrum of pharmacological activities. However, their specific efficacy against lung cancer remains largely unexplored and warrants further investigation. The appropriate isolation of GELNs is fundamental to all related research, yet a systematic comparison of different extraction methods is currently lacking. This study aimed to evaluate the differences among GELNs extracted by various methods and to investigate their anti-lung cancer pharmacological activities. Methods: The study employed four common isolation methods-ultracentrifugation (UC), sucrose gradient UC (sgUC), membrane filtration, and polyethylene glycol-based precipitation (PEG-based precipitation) - to isolate GELNs. The GELNs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and zeta potential measurements. Stability was evaluated under various conditions, including saline, serum, and different storage temperatures. The compositional profiles of GELNs extracted by four methods were explored using non-targeted metabolomics. A549 cells and PC-9 cells were used to assess the cellular uptake and anti-lung cancer efficacy of the four GELNs types. Network pharmacology, molecular docking, and molecular dynamics simulations were integrated to elucidate the potential mechanisms underlying their anti-lung cancer effects. Results: The four methods successfully isolated GELNs with distinct profiles: UC achieved the highest protein yield (1.630 ± 0.022 g/kg), membrane filtration yielded the highest particle concentration (46.9 ± 6.71×10 Conclusion: This study demonstrates that although all four methods can isolate GELNs, UC is recommended for fundamental research due to its high protein yield, excellent stability, and potent in vitro anti-lung cancer activity. Furthermore, the anti-lung cancer activity of GELNs may be attributed to the regulation of GSK3B, PGR, and SRC by 10-Gingerol, Hexahydrocurcumin, and [6]-Dehydrogingerdione.