A comprehensive evaluation of stability and safety for HEK293F-derived extracellular vesicles as promising drug delivery vehicles

HEK293F-derived extracellular vesicles (HEK293F-EVs) have great potential as next-generation drug delivery vehicles. A comprehensive understanding of their batch stability and in vivo safety is prerequisite for clinical translation. HEK293F-EVs were purified using ultracentrifugation combined with size exclusion chromatography, and their physicochemical properties, such as morphology, size distribution, and biomarkers, were thoroughly characterized. Raman spectroscopy and multi-omics analyses were employed to elaborate their molecular composition. Blood kinetics and biodistribution were assessed via IVIS spectrum imaging. Additionally, long-term in vivo safety was evaluated following multiple-dose administration through hematology, serum biochemistry, cytokine/chemokine profiling, and histopathology. HEK293F-EVs exhibited stable yields, purity, physicochemical properties (morphology, size, zeta potential, and marker proteins), and chemical composition across different cell passages (P10, P20, P30), with no significant variations. Content profiling, including protein, miRNA, metabolite, and lipid, confirmed consistent molecular stability across five production batches. GO, Reactome, and KEGG analyses revealed minimal enrichment in pathways related to acute immune response or cytotoxicity. Blood kinetics studies indicated rapid clearance of HEK293F-EVs from circulation, though slightly slower than PEG-Liposomes. Organ biodistribution was comparable between HEK293F-EVs and PEG-Liposomes, with HEK293F-EVs potentially having longer retention times. Importantly, HEK293F-EVs exhibited a favorable preclinical long-term safety profile, showing low immunogenicity and fewer tissue lesions compared to PEG-Liposomes. Our study demonstrates that HEK293F-EVs maintain stable physicochemical characteristics and compositions across batches and possess a superior safety profile, suggesting their significant potential as a safe and reliable drug delivery platform for clinical applications.