Controlling the stability of monodisperse phospholipid-coated microbubbles by tuning their buckling pressure

Hypothesis: Monodisperse phospholipid-coated microbubbles, with a size and resonance frequency tuned to the ultrasound driving frequency, have strong potential to enhance sensitivity, efficiency, and control in emerging diagnostic and therapeutic applications involving bubbles and ultrasound. A key requirement is that they retain their gas volume and shell material during physiologic pressure changes and withstand the overpressure during intravenous injection. The shell typically comprises a mixture of a phospholipid (e.g., DSPC) mixed with a PEGylated phospholipid (e.g., DPPE-PEG5000). We hypothesize that (i) lipid-coated microbubbles destabilize when shell buckling occurs under pressurization, (ii) the overpressure at which buckling occurs (buckling pressure) is linked to the molar fraction of PEGylated lipid in the shell, and (iii) PEGylated lipid can be selectively expelled from the shell by fluidizing it at elevated temperatures. Experiments: The buckling pressure was measured using ultrasound attenuation spectroscopy while the ambient pressure was varied. When the ambient pressure increased, the microbubble resonance frequency dropped sharply due to shell buckling and the associated loss of elasticity. The buckling pressure P_b was obtained for monodisperse microbubbles formed by microfluidic flow-focusing, with DPPE-PEG5000 mixed with DSPC at molar fractions from 1.5% to 10%. Additionally, P_b was quantified for microbubbles containing 10 mol% PEG after heating at temperatures ranging from 40 ^∘C to 70 ^∘C. The molar PEG content of the microbubbles was analyzed using high-performance liquid chromatography. Findings: Quasi-static compression of a microbubble above its buckling pressure leads to its destabilization. Lowering the PEG molar fraction from 10 to 1.5% increased the buckling pressure from 3 kPa to 27 kPa. Similarly, heating the 10 mol% bubble suspension at 60 ^∘C for one hour raised the buckling pressure by 20 kPa, due to the selective loss of PEGylated lipid from the shell, without affecting the monodispersity of the bubbles. The higher buckling pressure significantly improved microbubble stability, allowing them to withstand pressurization cycles of up to 45 kPa, nearly three times the systolic blood pressure in vivo.