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

Area of Science:

• Plant Biotechnology
• Biochemistry
• Extracellular Vesicle Research

Background:

• Extracellular vesicles (EVs) are crucial for intercellular communication.
• Aloe vera gel is a potential source of bioactive compounds, including EVs.
• Efficient isolation of EVs from complex plant matrices like Aloe vera remains challenging.

Purpose of the Study:

• To compare four distinct protocols for isolating extracellular vesicles (EVs) from Aloe vera gel.
• To determine the optimal method for maximizing EV yield and purity.
• To evaluate the impact of cellulase digestion on EV extraction efficiency.

Main Methods:

• Four EV isolation protocols were tested: conventional ultracentrifugation (UC), UC with cellulase digestion, a hybrid method (cellulase, filtration, UC), and a method integrating cellulase with the EXODUS technique.
• EVs were characterized using BCA protein quantification, transmission electron microscopy (TEM), and nanoflow cytometry.
• Morphological analysis confirmed intact lipid bilayers and sizes ranging from 50-100 nm for all isolated EVs.

Main Results:

• Cellulase-assisted protocols (B, C, D) significantly improved particle recovery and purity compared to conventional UC (A).
• No substantial differences in particle concentration or purity were observed among the three cellulase-assisted methods (B, C, D).
• Conventional UC (A) yielded lower particle recovery and a higher protein-to-particle ratio.

Conclusions:

• Enzymatic degradation of the Aloe vera matrix using cellulase is critical for enhancing EV recovery and purity.
• Cellulase-assisted isolation methods offer superior performance over direct ultracentrifugation for Aloe vera EV extraction.
• These findings provide a foundation for optimized EV isolation from plant-based sources.