fig7

Figure 7. Preparation of magneto-EV and characterization of purified magneto-EVs. (A) Schematic illustration of the preparation of SPIO his-tag (SPIO-His), by conjugating hexahistidine (6 × His-tag) polypeptide to the carboxyl groups of SPIO particles using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (sulfo-N-hydroxysuccinimide) chemistry. (B) As a result of the high affinity between the His-peptide and nickel ion, the SPIO-His particles bind to Ni²⁺ immobilized on beads (e.g., Ni-NTA resins) for further purification. (C) Schematic illustration of the encapsulation of SPIO-His into EVs by electroporation and subsequent purification by removing unencapsulated SPIO-His from the elute using Ni-NTA affinity chromatography. (D) Size distribution as measured by dynamic light scattering (DLS) for SPIO-His, EVs, SPIO-His/magneto-EV/EV mixtures after electroporation, and the final purified elute, respectively. (E) TEM images of EV, SPIO-His and EV-SPIO, respectively. (F) Concentrating magneto-EVs using a magnet. Eluted magneto-EV solution was placed on a magnet overnight to pellet magneto-EVs. The photograph shows the pelleted magneto-EV at the bottom of a microcentrifuge tube. (G) T2-weighted (T2w) images of magneto-EVs at different concentrations, unlabeled EVs, and PBS. Mean R2 values of magneto-EVs are plotted with respect to their concentration, from which the r2 (relaxivity) was estimated. Reprinted with permission from^[18].