Bone Marrow Mesenchymal Stem Cell-Derived Exosomes and Exosome-Liposome Hybrids as Nanocarriers for Delivering Cas9-GFP Plasmid to Embryonic Kidney Cells

Exosomes are natural membrane-enclosed nanovesicles (30–150 nm) involved in cell-cell communication. Recently, they have garnered considerable interest as nanocarriers for the controlled transfer of therapeutic agents to cells. Here, exosomes were derived from bone marrow mesenchymal stem cells (BM-MSCs) using three different isolation methods, and the size exclusion chromatography (SEC) led to the isolation with the highest purity. The limitation of using pure exosomes for encapsulating large nucleic acids was then addressed by the development of exosome-liposome hybrids. The efficiency of these hybrids to transfer the Cas9-green fluorescent protein plasmid (pCas9-GFP) into the human embryonic kidney 293T (HEK293T) cells were evaluated compared to the pure exosomes. The method of combining exosomes with liposomes (freeze-thaw vs. direct mixing) was proved to be more decisive in determining the size of the vesicular hybrids. In contrast, the ionic nature of the liposomes (lipoplex vs. cationic) in these hybrids was proved to be more important in determining the transfection efficiency. Both Cas9-GFP-loaded exosomes and exosome-liposome hybrids were taken up well by the HEK293T and were able to transfect them with their plasmid loads; however, the pure exosome and then the vesicular hybrids incorporating lipoplex transfected the cells more efficiently than those containing cationic liposomes.