Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase

Background This study aimed to evaluate the potential of plasma-derived extracellular vesicles (EVs) as drug delivery carriers by employing two drug-loading techniques: coincubation and freeze‒thaw cycles. Methods EVs isolated via the polyethylene glycol (PEG) precipitation method were characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The size of the particles was 200.1 ± 66.6 nm. The isolated vesicles were loaded with 1000 µg/ml snake venom L amino acid oxidase (SVLAAO) via the coincubation method and subjected to freeze‒thaw cycles to prepare a novel formulation. The encapsulation efficiency (EE) of the loaded EVs was analysed at 30 and 60 minutes, and in vitro drug release profiles were evaluated for both methods and kinetic model for the same was determined. Results The coincubation method achieved an EE of 58.08 ± 0.060% after 60 minutes, which was greater than that of the freeze‒thaw method (55.80 ± 0.060%). Drug release studies demonstrated that 93% of the drug was released in 8.5 hours by the coincubation method, whereas the freeze‒thaw method resulted in faster release (99% in 6.5 hours) due to membrane disruption. The best fit value (R²) was highest for zero order kinetics model. Conclusion In conclusion, the coincubation method preserves EV membrane integrity, enabling sustained drug release, making it a promising strategy for targeted drug delivery applications. This study highlights plasma-derived EVs as innovative carriers for therapeutic delivery.