The objective of this study was to determine if plas-ma membrane vesicles (PMVs) could be exploited for effi-cient transfer of macro-biomolecules and mitochondria.PMVs were derived from mechanical extrusion, and madefusogenic (fPMVs) by incorporating the glycoprotein G ofvesicular stomatitis virus (VSV-G). Confocal microscopy ex-amination revealed that cytoplasmic proteins and mitochon-dria were enclosed in PMVs as evidenced by tracing withcytoplasmically localized and mitochondria-targeted EGFP,respectively. However, no fluorescence signal was detectedin PMVs from cells whose nucleus was labeled with anEGFP-tagged histone H2B. Consistently, qRT-PCR measure-ment showed that mRNA, miRNA and mitochondrial DNAdecreased slightly; while nuclear DNA was not measureable.Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclearproteins were barely detecsle. In addition, fPMVs carryingcytoplamic DsRed proteins transduced about ~40 % of recip-ient cells. The transfer of protein was further confirmed byusing the inducible Cre/loxP system. Mitochondria transferwas found in about 20 % recipient cells after incubation withfPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) weregenerated and cultivated with fPMVs. Cell enumeration dem-onstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control.Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane poten-tial in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria byusing fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases

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• Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria