Comparative evaluation of isolation protocols for Aloe vera-derived extracellular vesicles

The primary objective of this investigation was to systematically evaluate four distinct protocols for extracting extracellular vesicles (EVs) from Aloe vera, aiming to establish a methodology that maximizes both yield and purity. Fresh Aloe vera gel served as the source material for EV isolation via four approaches: (A) Conventional ultracentrifugation (UC); (B) UC preceded by cellulase digestion; (C) a hybrid approach involving cellulase treatment, membrane filtration, and subsequent UC; and (D) cellulase pretreatment integrated with the EXODUS technique. Characterization of the harvested vesicles was performed using BCA protein quantification, transmission electron microscopy (TEM), and nanoflow cytometry. Morphological analysis confirmed that EVs obtained through all four strategies possessed intact lipid bilayers with diameters ranging from 50 to 100 nm. However, protocol (A) resulted in significantly lower particle recovery and exhibited an elevated protein-to-particle ratio relative to protocols (B) (C), and (D). Statistical analysis revealed no substantial variations in particle concentration or purity among methods (B) (C), and (D). Collectively, these findings indicate that enzymatic degradation of the Aloe vera matrix using cellulase prior to isolation markedly enhances EV recovery and purity. Compared to direct ultracentrifugation, cellulase-assisted methods yield higher particle counts, minimize co-isolated contaminants, and produce a more homogeneous size distribution.