As reported in previous studies, we have prepared RSL3@O2-ICG NBs using the thin-film hydration ultrasound method.44 (link) First, mix DSPC and DSPE-PEG-2000 at a mass ratio of 9:1, and then add 10ul of 5 mM RSL3 in methylene chloride and methanol (2:1, vol/vol). After entirely dissolving and mixing, transfer the above solution to a beaker and evaporate to form a lipid film. The dried film was hydrated into a lipid suspension with 5mL of 100ug/mL ICG in PBS. Next, the suspension was extruded 20 times through a 200nm membrane through a micro-extruder (Avanti Polar Lipids, Alabaster, AL). Then, the squeezed suspension was transferred to a sealed vial, and the syringe was evacuated and replaced with pure oxygen. The mixture was then mechanically shaken in a dental mixer (YJT Medical Apparatus and Instruments, Shanghai, China) for 60 seconds and resuspended in 2 mL of PBS solution for storage at 4°C. All processes are carried out in a dark environment. The blank nanobubbles (without RSL3 or ICG) were prepared using the same method. The stability of the RSL3@O2-ICG NBs and blank nanobubbles was evaluated by a hemocytometer at different time points, such as 6 h, 12h, 24h, and 48h after preparation at 4°C.