Fabrication and Characterization of Plasma-Loaded Microbubbles

PMBs were fabricated by a modified emulsification process as described in previous study [23 (link)]. The mixture of Span 60, NaCl, Tween 80, PBS and polyethylene glycol (PEG-4000) was stirred at room temperature and autoclaved at 121℃ for 12 min, subsequently cooled down to 40 ℃. As Fig. 1 shows, 18 mL of the suspension was sonicated for 2 min with constant purging of plasma gas for 6 s. After standing still for 3 h and separating into 3 layers, 4 mL of the middle layer were diluted in 8 mL PBS. And the total mixture was sonicated again to obtain the PMBs.Fig. 1

Schematic illustration of the preparation and characterization of plasma loaded microbubbles (PMBs)

Then, the PMBs were evaluated by a light microscopy to determine the concentration with blood cell counting plate. The morphology characterization of PMBs was determined by a bright field microscope. The size distribution was measured at different time points for stability assessment using a multi-angle particle size analyzer (Brookhaven, USA). The key reactive species, nitric oxide (NO) and hydrogen peroxide (H₂O₂) existed in PMBs suspension and release levels after ultrasound sonication were measured with a corresponding assay kit according to the manufacturer’s instructions (Beyotime, China), respectively. The values for the control group were obtained with PBS solution.