Stability Challenges and Engineering Strategies of Plant-Derived Extracellular Vesicle-Like Particles: A Translational Perspective

Plant-derived extracellular vesicle-like particles (PD-EVLPs), composed of a phospholipid bilayer enriched with lipids, proteins, metabolites, and RNA, have emerged as promising therapeutic agents with intrinsic bioactivity (eg, anti-inflammatory and wound healing activities) as well as biocompatible nanocarriers. However, before their clinical translation, a key challenge lies in ensuring their stability in biological environments and achieving effective accumulation at disease sites. On one hand, the lack of standardized isolation protocols and preservation strategies where non-standardized methods compromise yield-purity balance, freeze-thaw cycles induce lipid rearrangements that lead to vesicle fusion, and lyophilization-induced ice crystals disrupt membrane integrity, poses a significant barrier to maintaining the dispersity, structural integrity, and purity of PD-EVLPs affecting their ex vivo stability. On the other hand, extreme conditions in gastric and intestinal fluids during administration can easily cause PD-EVLPs to degrade and their content to leak, while in vivo circulation necessitates avoiding rapid immune clearance in order to accumulate at target sites, further challenging their stability and bioavailability. In this review, we comprehensively evaluate the entire PD-EVLPs pipeline from preparation to application, dissecting potential stability concerns at each stage, including isolation, storage conditions, structural composition, and administration routes. We also explore the contribution of engineering strategies to enhancing PD-EVLPs stability while considering potential risks associated with these modifications. By establishing a comprehensive framework, we aim to provide concrete guidance for standardizing PD-EVLPs preparation protocols and designing preclinical studies, thereby streamlining their translation from bench to bedside.