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-β1/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 α-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.