Targeting Fibrotic Scars with Extracellular Vesicles Extracted from Mature Aloe vera Gel: Enhanced Antioxidant, Anti-inflammatory, and Antifibrotic Activity through M2 Macrophage Polarization and Inhibition of Myofibroblast Differentiation

Abstract

Background: Abnormal scarring and fibrotic skin disorders arise from dysregulated wound healing. While Aloe vera is widely recognized for its therapeutic properties, the potential of its extracellular vesicles (Av-EVs) remains underexplored. Objective: To isolate and characterize Av-EVs and evaluate their antioxidant, anti-inflammatory, and antifibrotic properties in vitro, focusing on extraction method and plant maturity. Methods: Av-EVs were isolated from the gel of mature and young Aloe vera leaves using manual (NB) or blender-based (B) homogenization and characterized by nanoparticle tracking analysis, transmission electron microscopy, protein quantification, and immunoblotting of EV-associated markers. Antioxidant and cytotoxicity assays (DPPH, alamarBlue) were followed by functional anti-inflammatory and antifibrotic analyses in LPS-stimulated THP-1 macrophages and TGF-{beta}1/vitamin C-activated human dermal fibroblasts (RT-qPCR, immunofluorescence). Proteomic analysis was performed to investigate maturity-dependent differences in Av-EV cargo. Results: Mature NB-derived EVs exhibited the strongest bioactivity, with superior antioxidant capacity, suppression of pro-inflammatory mediators, enhanced M2 macrophage polarization, and significant downregulation of COL1A1 and -SMA. Immunoblotting further supported Av-EV identity and sample purity. B-derived and young EVs showed reduced bioactivity, with the latter failing to inhibit fibrotic markers. Conclusion: Manually extracted Av-EVs from mature leaves demonstrate superior multifunctional bioactivity, supporting their potential as plant-derived nanotherapeutics for fibrotic scar modulation.