Safety Evaluation of Long-term Administration of Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicles

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) hold significant therapeutic promise in regenerative medicine; however, their long-term safety profile remains inadequately characterized for clinical translation. In this study, human umbilical cord MSC-derived EVs (hucMSC-EVs) were produced using a heterologous component-free platform by substituting foetal bovine serum (FBS) with human platelet lysate (hPL). A comprehensive safety assessment was conducted by benchmarking hucMSC-EVs against polyethylene glycol (PEG)-liposomes. Pharmacokinetic analysis via cell membrane red fluorescent probe (DID)-labelling revealed rapid clearance of hucMSC-EVs, with a blood half-life of approximately 3 hours and predominant hepatic accumulation. Multi-dose evaluations integrating hematological profiling, cytokines/chemokines quantification, and histopathological scoring demonstrated superior biocompatibility of hucMSC-EVs over PEG-liposomes. Key findings include: 1. hucMSC-EVs did not induce activation of blood inflammatory cells; 2. hucMSC-EVs caused a significant increase in CCL12 only, and the rest of the multiple inflammatory factors/chemokines remained stable; 3. histopathological changes associated with hucMSC-EVs were mainly localized to liver and lungs, yet markedly less severe than PEG-liposome-induced lesions; 4. Transcriptome sequencing further demonstrated that hucMSC-EVs regulated metabolism and complement-coagulation cascade while avoiding activating pro-inflammatory pathways. In contrast, PEG-liposomes up-regulated inflammation-associated pathways such as IL-17 signalling, the MAPK pathway, the TLR pathway, and the T-cell differentiation pathway. This study establishes a multidimensional safety evaluation framework, providing critical preclinical evidence to advance the clinical translation of hucMSC-EVs