Isolation of MSC-Derived Exosome for Encapsulation of Doxorubicin

Background and Aim: Exosomes as natural nanoparticles are extracellularmembrane-derived vesicles with a 30-120 nm in diameter. Exosomes canbe derived from any different cells type. Among these different cells type,MSCs have several features such as the large ex vivo expansion capacitythat make them perfect candidates as a manufacturer of exosomes fordrug delivery. In this regard, it was demonstrated that MSC-derivedexosome can be loaded with anticancer drugs in order to increase theirtherapeutic index.Methods: In the current study, mouse MSCs was isolated from the adultmice bone marrow tissue. MSCs was cultured in DMEM supplementedwith 10% FBS and penicillin/streptomycin. MSCs were used at passage3-4 for exosome preparation. The proliferated MSCs (with 7% confluency)were cultured in DMEM/F12 supplemented with 10% exosome-depletedFBS and 1% penicillin-streptomycin for 72 h. Then, the exosomes fromexosome-depleted culture medium were isolated using ExoQuick-TC kit.The exosome pellets were re-suspended in phosphate buffered saline(PBS) and quantification of exosome was performed through Bradfordmethod. For drug loading, DOX-exosome mixture was sonicated using aModel 505 Sonic Dismembrator with .25″ tip with the following settings:20% amplitude, 6 cycles of 30 s on/off for three minutes with a 2-minutecooling period between each cycle. After sonication, the exo-DOXsolution was incubated at 37°C for 60 min. At the final stage, we useddynamic light scattering (DLS) and atomic force microscopy (AFM) forcharacterization of exosomes.Results: Obtained data indicated that the MSC-derived exosomes are 70–90 nm in diameter using DLS and AFM analysis. It was demonstrated thatMSC-derived exosomes were successfully purified. Exosome suspensionswere kept at -20°C temperature for one year. Based on the data from theBradford method, the Quantification of exosome was about 200 μg/mL.We applied UV-spectroscopy to determine the loading capacity of DOX.The amount of DOX-loaded into exosomes was 90% approximately. DLSstudies revealed that the size of exo-DOX nanoformulations increasedafter drug loading and became 140 nm.Conclusion: MSC-derived exosomes are the best choice as thenanovehicle for drug delivery by overcoming various biological barriers.In order to use in the clinic, there were several limitations. Includingthe efficient loading exosomes with a therapeutic agent without primarychanges in the content and structure of exosomal membranes and thesize of these. While the use of exosomes as vehicle therapeutic drugs isstill in its early stages, the potential for targeted drug delivery has beenclearly confirmed.

کلیدواژه ها:

Exosome ، Doxorubicin ، Anticancer ، Mesenchymal stem cell