Plant-derived exosome-like nanoparticles (PELNPs) represent promising bioactive agents, with ginger-derived exosome-like nanoparticles (GELNPs) exhibiting notable therapeutic potential due to their anti-inflammatory, antioxidant, and neuroprotective properties. While polyethylene glycol (PEG)-based extraction offers a costeffective alternative to ultracentrifugation for PELNP isolation, methodological refinements are needed to enhance yield, stability, and reproducibility. This study systematically optimizes GELNP isolation by integrating PEG6000 precipitation with pH adjustment and 0.45 µm filtration, building upon existing ginger nanovesicle protocols. We present the first comparative evaluation of pH modulation versus filtration, demonstrating distinct trade-offs: pH-adjusted GELNPs achieved smaller size, whereas filtered GELNPs exhibited superior colloidal stability and lower polydispersity index (PDI). Furthermore, we identify previously unreported seasonal variations in GELNP properties, with July-derived nanoparticles displaying enhanced stability and reduced size compared to those isolated in other months. Comprehensive physicochemical characterization—including transmission electron microscopy (TEM), atomic force microscopy (AFM), particle size analysis (PSA), zeta potential analysis, and Fourier-transform infrared spectroscopy (FT-IR)—confirmed differences in morphology, size distribution, surface charge, and biomolecular composition. These findings highlight the necessity for seasonally adjusted, optimized isolation protocols to ensure batch-to-batch consistency and maximize therapeutic efficacy, particularly for drug delivery applications. By addressing critical methodological gaps and introducing temporal considerations, this work establishes a framework for scalable, high-quality GELNP production.

Keywords