(M1130-06-40) Extraction, Characterization and Biodistribution of Plant-Derived Exosomes for Drug Delivery

Purpose: Plant-derived extracellular vesicles or exosomes are cell-derived lipid membrane-bound nanoparticles with emerging potential as drug delivery system due to their unique biocompatibility, low immunogenicity, and minimal toxicity. This study aimed to isolate, characterize, and evaluate the biodistribution of plant-derived exosomes in mice after intravenous (IV) administration.

Methods: Exosomes were isolated from no-GMO plant seeds through ultracentrifugation. Physicochemical properties and stability were determined by dynamic light scattering. Protein concentration and particle numbers were measured via BCA and nanoFCM analysis and morphology was determined by cryoEM. Cellular uptake in human SK-BR-3 cells was evaluated using PKH26-dye labeled exosomes and fluorescence microscopy. Radiotracer labeled ³H-CHE-exosomes containing ³H-cholesteryl hexadecyl ether were prepared via sonication and purified by gel filtration column. Female CD-1 mice (6-8 weeks, N=4) received ³H-CHE-exosomes at a dose of 1μCi/25g of mouse via IV. Serial blood samples were collected at 8 time points within 24 h and major organs were harvested at 24 h post injection for radioactivity analysis using liquid scintillation counting.

Results: Exosomes had a mean particle size of 283±66 nm, zeta potential of -56.9±1.7 mV, and polydispersity index of 0.15. CryoEM confirmed spherical morphology. Exosomes contained 3±2μg protein on 1×10⁹ particles and yielded 6.9 ×10¹³ ±5.6×10¹² particles/g of seeds. Exosomes remained stable at -80°C for 5 months, but were prone to aggregate at 4°C storage condition after 3 months. Fluorescent dye PKH26 labeled exosomes were efficiently internalized by human SK-Br-3 cells after 7 h of incubation. After IV administration in mice, ~70% of injected dosed (%ID) of the radiolabeled exosomes localized to the reticuloendothelial organs including liver and spleen with minimal distribution to heart, kidneys, muscles, and brain 24 h post injection. Mass balance showed 46% of total injected dose was recovered from blood and liver, spleen, lung, heart, kidneys and brain at 24 h post injection.

Conclusion: Plant seed-derived exosomes were successfully isolated and characterized. The nanosized exosomes were stable, can be internalized by human cells efficiently, and showed promising biodistribution in mice following IV administration.