A Step-by-Step Protocol for the Isolation of Aloe vera-Derived Extracellular Vesicles via Manual and Shear-Force...

Aloe vera has long been used for its diverse pharmacological properties, motivating continued interest in isolating and preserving the bioactive molecules responsible for its therapeutic potential. More recently, Aloe vera-derived extracellular vesicles (Av-EVs) have emerged as nanoscale, cell-free carriers capable of retaining and delivering these properties, making them attractive for various biomaterials, nanomedicine, and regenerative medicine applications. Multiple techniques are available for extracellular vesicle isolation. These include ultracentrifugation, polymer-based precipitation, size-exclusion chromatography, immunoaffinity capture, ultrafiltration, density gradient separation, and emerging microfluidic platforms. Each method presents distinct trade-offs in purity, yield, scalability, and downstream compatibility. Despite this diversity, standardized workflows tailored to Av-EV isolation remain limited, and the influence of homogenization-induced shear forces and plant maturity on vesicle recovery and characterization has not been systematically addressed. Here, we present a reproducible protocol for isolating Av-EVs from Aloe vera gel employing two distinct homogenization strategies: manual, no-shear force (NB EVs), and blender-based shear-force homogenization (B EVs). The workflow covers gel preparation, serial centrifugation for debris removal, ultracentrifugation as the gold standard for vesicle enrichment, and final sterile filtration. This protocol enables consistent recovery of Av-EVs suitable for physicochemical characterization and functional analyses. It is simple and relies on commonly available laboratory equipment, facilitating broad adoption by ultracentrifugation users and offering adaptability to diverse research projects involving purified Aloe vera gel and Av-EVs, including studies focused on wound healing, fibrotic scarring, and regenerative processes, where coordinated antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and moisturizing responses are of interest. Key features • This protocol allows direct comparison of vesicle yield, size distribution, and protein content across extraction methods. • This protocol yields ~1.4-2.0 × 10¹⁰ particles/mL per mature leaf for a total of ~8 × 10¹² particles per leaf. • This protocol yields ~1.2-2.8 × 10¹⁰ particles/mL per young leaf for a total of ~2.8 × 10¹² per leaf. • EVs from mature Aloe leaves yield protein concentrations of ~160-447 μg/mL, corresponding to ~3,840-10,728 μg of protein per leaf.