Echogenic exosomes as ultrasound contrast agents

Abstract

Exosomes are naturally secreted extracellular bilayer vesicles (diameter 40–130 nm), which have recently been found to play a critical role in cell-to-cell communication and biomolecule delivery. Their unique characteristics—stability, permeability, biocompatibility and low immunogenicity—have made them a prime candidate for use in delivering cancer therapeutics and other natural products. Here we present the first ever report of echogenic exosomes, which combine the benefits of the acoustic responsiveness of traditional microbubbles with the non-immunogenic and small-size morphology of exosomes. Microbubbles, although effective as ultrasound contrast agents, are restricted to intravascular usage due to their large size. In the current study, we have rendered bovine milk-derived exosomes echogenic by freeze drying them in the presence of mannitol. Ultrasound imaging and direct measurement of linear and nonlinear scattered responses were used to investigate the echogenicity and stability of the prepared exosomes. A commercial scanner registered enhancement (28.9% at 40 MHz) in the brightness of ultrasound images in presence of echogenic exosomes at 5 mg mL⁻¹. The exosomes also showed significant linear and nonlinear scattered responses—11 dB enhancement in fundamental, 8.5 dB in subharmonic and 3.5 dB in second harmonic all at 40 μg mL⁻¹ concentration. Echogenic exosomes injected into the tail vein of mice and the synovial fluid of rats resulted in significantly higher brightness—as much as 300%—of the ultrasound images, showing their promise in a variety of in vivo applications. The echogenic exosomes, with their large-scale extractability from bovine milk, lack of toxicity and minimal immunogenic response, successfully served as ultrasound contrast agents in this study and offer an exciting possibility to act as an effective ultrasound responsive drug delivery system.