Background

Plant-derived extracellular vesicles (PDEVs) have emerged as a promising class of naturally occurring nanovesicles that retain the therapeutic benefits of the parent plant while functioning as biocompatible carriers of lipids, proteins, nucleic acids, and metabolites. Delivery of this molecular cargo supports intercellular communication, maintenance of tissue homeostasis, and regenerative processes [1]. Among PDEVs, Aloe vera–derived extracellular vesicles (Av-EVs) have attracted particular attention owing to the plant’s well-recognized pharmacological properties. Aloe vera contains a rich repertoire of bioactive molecules that collectively confer wound healing, antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and moisturizing effects [2–7]. Historically, these constituents have motivated extensive investigation of Aloe vera–based topical and oral formulations, with numerous in vitro studies, preclinical models, and clinical trials demonstrating high efficacy in skin-related applications [8–25]. Although such formulations remain widely used, recent interest has shifted toward Av-EVs, in which these bioactive molecules are enriched within a small vesicular fraction (~1% of the total gel volume) rather than being dispersed throughout the predominantly aqueous matrix [5].

Preliminary studies indicate that Av-EVs can modulate inflammatory signaling, enhance antioxidant defenses, and inhibit myofibroblast differentiation and contractile activity. Their versatility has also been demonstrated in applications such as drug encapsulation for targeted cancer therapy [26–29]. However, reported isolation methods vary widely, and to our knowledge, no prior study has provided a standardized workflow tailored to Av-EV isolation or systematically examined the combined influence of homogenization-induced shear forces and plant maturity on physicochemical characterization and functional analyses.

Multiple techniques have been described for the isolation of extracellular vesicles, including ultracentrifugation, polymer-based precipitation, size-exclusion chromatography, immunoaffinity capture, ultrafiltration, density gradient separation, and emerging microfluidic platforms. Each method presents distinct trade-offs in terms of purity, yield, scalability, and instrumentation requirements [30]. In this protocol, ultracentrifugation was selected as the gold-standard strategy due to its widespread adoption, reproducibility, and compatibility with downstream analyses. Here, we describe a reproducible workflow for isolating Av-EVs that incorporates two homogenization strategies: manual, no-shear force (NB EVs), and blender-based shear-force homogenization (B EVs). These steps are followed by serial centrifugation for debris removal, ultracentrifugation for vesicle enrichment, and final sterile filtration.

By enabling controlled comparison across extraction strategies and plant maturity stages, the protocol enables systematic evaluation of how processing conditions and source material impact Av-EV properties. The resulting vesicles can then be employed in experimental contexts related to pathological wound healing, such as fibrotic scarring, where coordinated antioxidant, anti-inflammatory, and pro-regenerative responses are relevant. In these settings, Av-EVs may contribute to restoring redox balance, modulating persistent inflammatory responses, and limiting excessive extracellular matrix deposition associated with fibrosis. In particular, our study demonstrated that the manual approach preserved vesicle integrity and bioactivity more effectively than shear-based homogenization, highlighting the importance of the extraction strategy for maintaining vesicle quality [31]. Additionally, EVs derived from mature leaves consistently outperformed those obtained from younger plants, showing stronger downregulation of inflammatory mediators and fibrotic markers [31]. Together, these findings suggest that both extraction strategies and plant maturity are crucial variables that must be carefully controlled to ensure the reproducibility and potency of EV-based therapeutics derived from Aloe sources.

While developed in the context of skin fibrosis research, this workflow can be broadly applied to studies investigating Av-EVs in biomaterials, nanomedicine, and regenerative medicine. For instance, future work may examine whether additional Aloe-associated properties, such as antimicrobial, analgesic, and moisturizing activities, are also mediated by its vesicles. Exploring these potential functions represents an important area for further broadening our understanding of the translational potential of Av-EVs in therapeutic delivery systems, bioactive materials, and tissue regeneration applications.