Microbubble Preparation via Vibration

Microbubbles were prepared using a simple mechanical vibration method.16 (link) Briefly, 5 mg of DPPC, 2 mg of DSPE, and 0.5 mg of CHOL were dissolved in 500 µL of 10% glycerol solution in a vial. After heating in a water bath at 50°C for 30 minutes, the air in the vial was exchanged with perfluoropropane (C₃F₈; Research Institute of Physical and Chemical Engineering of Nuclear Industry, Tianjin, China), followed by vigorous shaking for 45 seconds via dental amalgamator (YJT; Shanghai Medical Apparatus and Instruments, Shanghai, China). Finally, the microbubbles were washed with PBS twice to obtain supernatant microbubbles via centrifugation at 500 rpm, and the concentration was then adjusted to 10⁸ per mL. The particle size and potential of the microbubbles were analyzed using a laser particle size analyzer system (Zetasizer 3000 HS; Malvern Instruments). The morphological characteristics of microbubbles were analyzed using an inverted optical microscope (IX71; Olympus, Tokyo, Japan).