Isolation of extracellular vesicles and their application in drug delivery

Extracellular vesicles (EVs) are nano-sized vesicles with a phospholipid bilayer that transport proteins, nucleic acids, and bioactive molecules, acting as key intercellular messengers. This thesis addresses the need for scalable, high-yield EV production using reproducible downstream processing methods, driving the exploration of alternative biological sources. Lemon juice was used as a model fluid to develop a scalable membrane-based process for EV isolation, achieving high yield and purity. The optimized tangential flow filtration (TFF) protocol outperformed ultracentrifugation (UC) and size exclusion chromatography (SEC), providing an efficient approach for large-scale EV production. The robustness of this method was further evaluated using milk whey-derived EVs, a valuable dairy by-product. These EVs were loaded with curcumin to enhance its therapeutic potential against neuroblastoma, overcoming its low bioavailability. The optimized TFF process enabled simultaneous EV purification and removal of unbound curcumin in a single step. The therapeutic efficacy of curcumin-loaded whey EVs was confirmed against neuroblastoma SK-N-AS cell lines, highlighting their potential as biocompatible delivery vehicles. Lemon-derived EVs were investigated for small interfering RNA (siRNA) delivery in oncogene silencing applications. Optimized siRNA loading ensured effective encapsulation and cellular uptake in target cells, validating their potential as a scalable, non-toxic gene therapy system. Stability testing confirmed that lemon-derived EVs retained structural integrity for six months under low-temperature and lyophilized conditions, supporting their viability for therapeutic applications. Additionally, sustainable methods for extracting silk proteins, sericin and fibroin, from Bombyx mori cocoons were developed using eco-friendly techniques, aligning with sustainable bioprocessing goals. This work advances scalable EV isolation techniques, underscores the therapeutic potential of plant- and dairy-derived EVs, and highlights sustainable biomaterials as innovative solutions for healthcare applications.