Abstract
Extracellular vesicles (EVs) have emerged as promising natural nanocarriers for therapeutic delivery due to their intrinsic biocompatibility, membrane stability, and ability to transport diverse
bioactive cargo. Among them, plant-derived EVs offer unique advantages, including scalability,
low immunogenicity, and the natural encapsulation of health-promoting phytochemicals.
However, their broader application has been limited by the lack of standardized, high-purity
isolation methods and incomplete understanding of their therapeutic potential. The overall
objective of this dissertation was to develop affinity-based technologies for the isolation of plant
derived EVs, characterize their biochemical cargo, evaluate their anticancer activity, and establish
their potential as engineered nanocarriers for targeted therapeutic delivery.
A universal affinity-based EV isolation platform was first developed using the phosphatidylserine
binding Lactadherin C1C2 domain fused to an intein–chitin binding domain (CBD), enabling
trace-free purification of EVs through intein-mediated self-cleavage. This platform was
successfully validated across multiple biological kingdoms, including mammalian cells, plants,
fungi, bacteria, and complex biological fluids, demonstrating efficient recovery of intact EVs while
minimizing contamination associated with conventional ultracentrifugation. The strategy was
subsequently adapted for the selective isolation of plant-derived EVs from Arabidopsis thaliana,
where affinity-captured vesicles exhibited reduced protein aggregate contamination while
preserving vesicle morphology, size distribution, and molecular integrity.
Comprehensive biochemical characterization confirmed that plant-derived EVs naturally
encapsulate glucoraphanin, a glucosinolate precursor of the anticancer compound sulforaphane.
Functional studies demonstrated selective inhibition of A549 lung cancer cell proliferation with
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minimal toxicity toward non-cancerous HEK293T cells and mesenchymal stem cells. Notably,
EVs isolated from Pseudomonas syringae-infected plants exhibited significantly enhanced
glucoraphanin loading and markedly greater anticancer activity than EVs isolated from healthy
plants. Myrosinase-mediated glucose release assays further confirmed that glucoraphanin
remained biologically active within the isolated EVs.
To further improve purification specificity, a second affinity platform based on engineered single
chain variable fragment (scFv) antibodies fused to an intein–CBD system was developed for
selective immunocapture of plant EV subpopulations from Arabidopsis, kale, and broccoli. This
approach enabled efficient enrichment of intact EVs with minimal non-vesicular contaminants
while preserving vesicle integrity and biological functionality. Immunomagnetic depletion of
TET8-positive EVs further demonstrated that the observed anticancer activity originated
specifically from plant-derived EVs rather than bacterial extracellular vesicles or protein
aggregates.
Collectively, this dissertation establishes robust affinity-based technologies for high-purity
isolation and characterization of plant-derived extracellular vesicles while demonstrating their
intrinsic therapeutic potential as natural carriers of glucoraphanin. These findings provide new
insights into plant EV biology and lay the foundation for the development of scalable,
biocompatible EV-based nanotherapeutics for targeted cancer treatment and future translational
applications.